Umbilical cord biomaterial for medical use

ABSTRACT

The present invention provides a biomaterial comprising a mammalian umbilical cord membrane. The biomaterial can additionally comprise Wharton&#39;s jelly and/or one or more umbilical cord vessels. The biomaterial is preferably dry, and can be flat, tubular, or shaped to fit a particular body structure. The invention further provides laminates comprising at least one layer of an umbilical cord membrane biomaterial. The invention further provides methods of making the biomaterial, and laminates comprising the biomaterial, and methods of using the biomaterial.

1. FIELD OF THE INVENTION

The present invention generally relates to biomaterials derived from theumbilical cord membrane, compositions comprising the umbilical cordmembrane, and methods of treatment using the compositions.

2. BACKGROUND OF THE INVENTION

The repair or treatment of various body tissues, such as skin, organs,and the like, has been accomplished using collagen compositions,including membranes comprising collagen. A need exists, however, foradditional such compositions, including ones that are able to handleloads well.

3. SUMMARY OF THE INVENTION

The present invention provides biomaterials derived from umbilical cord.In certain embodiments, the biomaterial comprises an umbilical cordmembrane. The present invention provides methods of making the umbilicalcord biomaterial, and of using the biomaterial, e.g., to repair organsand tissues.

In one aspect, the present invention provides a biomaterial comprisingan isolated mammalian umbilical cord membrane. In a specific embodiment,the biomaterial comprises at least one umbilical vessel (e.g., anumbilical artery or umbilical vein). In another specific embodiment, thebiomaterial comprises Wharton's jelly. In another specific embodiment,the biomaterial comprises Wharton's jelly but lacks umbilical vessels.In another specific embodiment, the biomaterial comprises the umbilicalcord membrane, Wharton's jelly, and all three umbilical vessels. Theumbilical cord biomaterial is not, however, an umbilical cord that hasnot been processed in any manner.

In more specific embodiments of any of the above embodiments, thebiomaterial comprises less water by weight than native umbilical cordmembrane in vivo, e.g., the biomaterial comprises 40%, 30%, 20%, 10% orless water by weight. In other more specific embodiments, thebiomaterial comprises at least 60%, at least 70%, or at least 80% waterby weight. In another specific embodiment, the biomaterial isdecellularized prior to use. In another specific embodiment, thebiomaterial is not decellularized prior to use. In another specificembodiment, the umbilical cord membrane of the biomaterial is cut orslit longitudinally. In another specific embodiment, the biomaterial issubstantially flat. In another specific embodiment, the biomaterial issubstantially tubular. In another specific embodiment, the biomaterialcomprises artificially crosslinked proteins.

In another specific embodiment, the umbilical cord biomaterial comprisesan exogenous bioactive molecule, that is, a bioactive molecule notobtained from the umbilical cord used to make the biomaterial. In a morespecific embodiment, said bioactive molecule is a cytokine or growthfactor. In another more specific embodiment, said bioactive molecule isan extracellular matrix protein. In another more specific embodiment,said extracellular matrix protein is collagen, fibronectin, elastin,vitronectin, or hyaluronic acid. In a more specific embodiment, saidbioactive molecule is hyaluronic acid. In an even more specificembodiment, said hyaluronic acid is crosslinked to said umbilical cordmembrane. In another specific embodiment, said biomaterial comprises anexogenous polymer. In a more specific embodiment, said exogenous polymeris a synthetic biodegradable polymer or an anionic polymer. In anothermore specific embodiment, said synthetic biodegradable polymer is apolyhydroxyalkanoate. In a more specific embodiment, said anionicpolymer is dextran sulfate or pentosan polysulfate. In another specificembodiment, the bioactive molecule is an antibiotic, a hormone, a growthfactor, an anti-tumor agent, an anti-fungal agent, an anti-viral agent,a pain medication, an anti-histamine, an anti-inflammatory agent, ananti-infective agent, a wound healing agent, a wound sealant, a cellularattractant or a scaffolding reagent. In another specific embodiment, thebioactive molecule is a small molecule, e.g., a small organic molecule,e.g., a drug.

In another specific embodiment, the umbilical cord biomaterial comprisesa hydrogel composition. In a more specific embodiment, said hydrogelcomposition comprises a polyvinyl alcohol, a polyethylene glycol, ahyaluronic acid, a dextran, or a derivative or analog thereof.

The umbilical cord biomaterial can also comprise, e.g., be seeded with,one or more types of stem and/or progenitor cells. In a specificembodiment, the biomaterial comprises an exogenous stem cell, that is, astem cell not native to the umbilical cord from which the biomaterial isderived, or from an individual different from the umbilical cord donor.In specific embodiments, the stem cell is a placental stem cell, amesenchymal stem cell, an embryonic stem cell, an adult stem cell, or asomatic stem cell. In a more specific embodiment, the somatic stem cellis a neural stem cell, a hepatic stem cell, a pancreatic stem cell, anendothelial stem cell, a cardiac stem cell, a stromal cell, or a musclestem cell. In another specific embodiment, the umbilical cordbiomaterial comprises an exogenous adult (e.g., fully-differentiated orcommitted progenitor) cell.

In another embodiment, the invention provides laminates comprising theumbilical cord biomaterial. Such laminates can comprise layers ofumbilical cord biomaterial, or one or more layers of umbilical cordbiomaterial layered with one or more layers of another material. Thelayers can be substantially aligned, or can be offset, e.g.,overlapping, to form, e.g., a laminate of biomaterial longer and/orwider than an umbilical cord. Thus, in one embodiment, the inventionprovides a laminate comprising a plurality (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, etc.) of layers, wherein at least one of the layers comprises anumbilical cord biomaterial. In a specific embodiment, said laminatecomprises one or more layers of an amniotic membrane material. In a morespecific embodiment, said amniotic membrane material is a dried amnioticmembrane laminated to said umbilical cord membrane. In another morespecific embodiment, said amniotic membrane material has been sonicatedor otherwise disrupted. In another specific embodiment, at least some ofthe proteins in at least one of the layers of the laminate areartificially crosslinked, either to other proteins in the same layer, orto proteins in one or more adjoining layers.

The invention further provides laminates comprising the umbilical cordbiomaterial and other useful compounds. For example, in one embodiment,the invention provides a laminate comprising umbilical cord biomaterialand an exogenous stem cell or an exogenous adult cell. In anotherembodiment, the laminate comprises a hydrogel composition. In a specificembodiment, said hydrogel composition comprises a polyvinyl alcohol, apolyethylene glycol, a hyaluronic acid, a dextran, or a derivative oranalog thereof.

The invention also provides a method of producing a biomaterialcomprising isolating and decellularizing a biomaterial comprising anumbilical cord membrane. In a specific embodiment, the biomaterialcomprises Wharton's jelly, or one or more umbilical cord vessels. Inanother embodiment, the method comprises isolating said umbilical cordmembrane from one or more umbilical cord vessels. The method may furthercomprise drying said biomaterial to less than 20% water by weight. Invarious embodiments, said biomaterial is dried at about 26° C. to about65° C., or at about 35° C. to about 50° C. The composition can, forexample, be dried with a hygroscopic compound. In another embodiment,the biomaterial is freeze dried, or dried using vacuum. Saiddecellularizing may, for example, comprise contacting the umbilical cordmembrane with a detergent solution, for example, a solution comprising0.01-2.0% deoxycholic acid. The detergent may be a nonionic detergent,an anionic detergent, or a combination thereof. In specific embodiments,said detergent is Triton X-100 or sodium dodecyl sulfate, or acombination thereof.

The invention further provides methods of making a laminate comprisingan umbilical cord membrane biomaterial. Such a method can comprisecrosslinking at least some proteins in at least one layer of saidlaminate. The method of making the laminate, in one embodiment,comprises layering a plurality of umbilical cord membranes in contactwith each other to form a laminate. In a specific embodiment, one ormore of said membranes comprise less than 20% water by weight prior tosaid layering. In another specific embodiment, said laminate is dried toless than 20% water by weight after said layering. In other specificembodiments, one or more of said membranes comprise at least 60%, atleast 70% or at least 80% water by weight. In another specificembodiment, said laminate comprises at least 60%, at least 70%, or atleast 80% water by weight.

The invention also provides for the use of the umbilical cord membranebiomaterial to deliver one or more therapeutic agents to a subject. Inone embodiment, the invention provides a method of delivering atherapeutic agent to a subject comprising contacting the subject with anumbilical cord membrane biomaterial, or laminate thereof, wherein saidcomposition or laminate comprises a therapeutic agent. In a specificembodiment, said subject is a human. In other specific embodiments, saidtherapeutic agent is an antibiotic, an anti-cancer agent, ananti-bacterial agent, an anti-viral agent, a vaccine, an anesthetic, ananalgesic, an anti-asthmatic agent, an anti-inflammatory agent, ananti-depressant, an anti-diabetic agent, an anti-psychotic, a centralnervous system stimulant, a hormone, an immunosuppressant, a musclerelaxant, or a prostaglandin.

The invention further provides a method of using an umbilical cordbiomaterial, e.g., one that comprises an isolated umbilical cordmembrane, in the repair of a tympanic membrane deformity, comprisingcontacting said tympanic membrane with such a biomaterial. In a specificembodiment, the deformity is a perforation, which may be, e.g., acentral perforation or a marginal perforation. The perforation may becaused by, e.g., trauma, or as part of a surgical procedure. In a morespecific embodiment, said contacting is sufficient to occlude theperforation. In another more specific embodiment, said perforation hasnot healed spontaneously within two months of developing theperforation. In another specific embodiment, said deformity is anatelectatic tympanic membrane, a deformity relating to a choleastoma, aretraction pocket or a deformity resulting from a tympanosclerosis.

The invention also provides for the repair of other deformities usingthe umbilical cord membrane biomaterial. For example, the inventionprovides a method of repairing a nasal septum having a perforation,comprising contacting said septum with a biomaterial comprising anumbilical cord biomaterial, e.g., one comprising an isolated mammalianumbilical cord membrane. In a more specific embodiment, said methodcomprises contacting cartilage within said septum with said biomaterial.

As used herein, “exogenous bioactive compound” means a moleculeintroduced into the biomaterial that has a detectable effect on one ormore biological systems. Examples of bioactive compounds are listed,without limitation, in Section 5.1.2, below.

As used herein, “substantially flat,” in reference to umbilical cordbiomaterial, means that the majority of the umbilical cord biomaterialis planar, but can comprise differences in thickness, whethernaturally-occurring or artificially-induced, e.g., natural variations inmembrane thickness, ridges, patterns, raised areas, warps, and the like,induced, e.g., during drying on a mesh.

As used herein, “substantially tubular,” in reference to umbilical cordbiomaterial, means that the majority of the umbilical cord biomaterialis tubular (i.e., circular, ovoid, or irregular in cross-section, andcomprising the material defining an interior or lumen). Thesubstantially tubular biomaterial can be a closed tube or partially opentube.

As used herein, “umbilical cord biomaterial” means any biomaterialcomprising an isolated umbilical cord outer membrane, particularly abiomaterial manufactured or derived from umbilical cord, and includes,e.g., dried whole umbilical cord, dried umbilical cord membrane (with orwithout vessels), umbilical cord membrane laminated with a secondmaterial, etc. The term does not, however, encompass an umbilical cordthat has not been treated or manipulated in any manner, that is, anumbilical cord that has not been modified from an in vivo state.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict the effect of radiation dose on water uptake [⋄,(Ww−Wd)/Wd*100] and equilibrium water content [□, (Ww−Wd)/Ww*100] forhuman umbilical cord biomaterial incubated for 10 (A) and 20 (B) days in1% D-cell solution.

FIGS. 2A and 2B depict a comparison of the change in thickness (□) andwater uptake (▪) during rehydration of gamma sterilized human umbilicalcord biomaterial incubated for 10 (A) and 20 (B) days in 1% D-cellsolution. Error bars indicate standard deviation.

FIG. 3 depicts a comparison of the denaturation temperature ofrehydrated human umbilical cord membrane that had previously beenincubated in 1% D-cell solution for 10 or 20 days. There is essentiallyno difference between the different incubation times and there is alinear decrease in denaturation temperature with increasing radiationdose.

FIG. 4 depicts a schematic of tensile testing of membrane samples basedon American Society for Testing and Materials (ASTM) standard D1708. Adog bone shaped sample 1 is mounted in a rectangular support of vellumpaper. The membrane and the support are incubated in PBS for 1 hour at37° C. The support keeps the membrane flat and eases loading into themechanical tester. The tester comprises and upper grip 2 and a lowergrip 3. Once the support and membrane have been secured in the grips ofthe mechanical tester with glue 4, the supporting struts 5 are cut andthe sample can be tested with no interference from the vellum support.

FIG. 5A depicts a suture pull-out assay apparatus with an lower grip 10holding vellum paper 11 to which a 1×2 cm section of umbilical cordbiomaterial 12 is glued, and an upper grip 13 attached to a suture 14that passes through the umbilical cord biomaterial. Force was appliedupwards through the suture at approximately 12.7 mm/min. FIG. 5B depictsresults of a comparison of the human umbilical cord biomaterial (HUC)and dried human amniotic membrane pull-out resistance in Newtons (N).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Umbilical Cord Biomaterial

5.1.1 Description

The umbilical cord biomaterial of the invention is derived from amammalian umbilical cord or part thereof that comprises an umbilicalcord membrane (that is, the outer membrane of the umbilical cord). Theumbilical cord is a substantially tubular organ, typically 10-15 cm inlength, that connects the fetus to the placenta and houses the umbilicalvessels. The umbilical cord comprises an outer membrane that wrapsaround two umbilical arteries and one umbilical vein, which arecontained within a ground substance known as Wharton's jelly. The maincomponents of Wharton's jelly are proteoglycans. Wharton's jelly alsocontains large, stellate fibroblasts and macrophages.

The umbilical cord membrane biomaterial of the invention typicallycomprises only the umbilical cord membrane, but can also compriseWharton's jelly and/or one or more of the umbilical vessels. In oneembodiment, the umbilical cord biomaterial is an umbilical cord membranesubstantially isolated from the remaining umbilical cord components(e.g., Wharton's jelly and umbilical vessels). In another embodiment,the umbilical cord biomaterial comprises an umbilical cord membrane andWharton's jelly (that is, the ground material in which the umbilicalcord vessels are contained in the intact umbilical cord) that areisolated from the remaining umbilical cord components (e.g., umbilicalcord vessels). In another specific embodiment, the umbilical cordmembrane biomaterial comprises the membrane, Wharton's jelly and one ormore umbilical cord vessels. In another embodiment, the umbilical cordbiomaterial comprises an isolated umbilical cord (e.g., comprisingWharton's jelly and vessels, Wharton's jelly only, or vessels only) thathas been flattened into a sheet or strip. The umbilical cord membranebiomaterial can be a substantially tubular structure from which thecontents (Wharton's jelly and vessels) have been removed. Thebiomaterial can also comprise an umbilical cord membrane that has beenslit or cut for part or all of the length of the umbilical cord toexpose the contents of the umbilical cord.

In a specific embodiment, the biomaterial, comprising umbilical cordmembrane and/or Wharton's jelly and/or vessels) can be decellularized.In another specific embodiment, the biomaterial comprises umbilical cordmembrane-associated cells or Wharton's jelly-associated cells that havebeen killed. In another specific embodiment, the biomaterial comprisesumbilical cord membrane-associated cells or Wharton's jelly-associatedcells that have been maintained in a living state.

The umbilical cord biomaterial of the invention can be derived from theumbilical cord of any mammal, for example, from equine, bovine, porcineor catarrhine sources, but is most preferably derived from humanumbilical cord.

The umbilical cord biomaterial is preferably dry or substantially dry.In a preferred embodiment, the umbilical cord biomaterial issubstantially dry, i.e., is 20% or less water by weight. When dry, theumbilical cord biomaterial can be substantially flat. The drybiomaterial may, in another embodiment, substantially retain the shapeof the native umbilical cord, that is, the dry membrane may besubstantially tubular. The umbilical cord biomaterial can also be shapedto assume different conformations, e.g., can be curved, cut, molded, orthe like, to fit to a part of the body.

In another preferred embodiment, the umbilical cord biomaterial has notbeen protease-treated, heat-denatured or artificially (e.g., chemicallyor radiologically) crosslinked. In another embodiment, the umbilicalcord biomaterial comprises artificially crosslinked proteins, e.g.,chemically or radiologically crosslinked collagen. In other embodiments,the umbilical cord biomaterial contains substantially no structuralproteins that are artificially crosslinked. For example, in oneembodiment, the umbilical cord biomaterial is not fixed. A preferredumbilical cord biomaterial is produced by the methods disclosed herein(see Section 5.1.4, below, and Examples 1 and 2).

When the umbilical cord biomaterial is substantially dry, it istypically about 0.001 g/cm² to about 0.006 g/cm². In a specificembodiment, a single layer of the acellular, dried umbilical cordbiomaterial is approximately 50 microns to 250 microns in thickness,typically approximately 90 microns to 220 microns in thickness. In otherspecific embodiments, a single layer of the umbilical cord biomaterialis approximately 75-200 microns, 100-200 microns, 100-220 microns,120-220 microns, or 150-250 microns in thickness in the dry state. Inanother embodiment, the average thickness of the umbilical cordbiomaterial is about 157 microns (e.g., ±20%). In another specificembodiment, the pull out strength of the dried umbilical cordbiomaterial is approximately 1.5 Newtons (N), compared to a pull outstrength of a dried amniotic membrane material at approximately 0.4N.

Generally, the umbilical cord biomaterial is sided, that is, theumbilical cord biomaterial comprises an epithelial side (from theinterior of the umbilical cord), and an outer, mesothelial side (fromthe exterior of the umbilical cord).

Generally, the umbilical cord biomaterial is non-immunogenic.

In various embodiments, the umbilical cord biomaterial comprisesparticular cytokines, i.e., interleukin (IL)-1b, IL-2, IL-3, IL-6, IL-7,IL-12, IL-15, IFN-α, MIP-1b, and/or MCP-1.

In one embodiment, the umbilical cord biomaterial is translucent. Inother embodiments, the umbilical cord biomaterial is opaque, or iscolored or dyed, e.g., permanently colored or dyed, using amedically-acceptable dyeing or coloring agent. Such an agent may beadsorbed onto the biomaterial, or the biomaterial may be impregnated orcoated with such an agent. In this embodiment, any known non-toxic,non-irritating coloring agent or dye may be used.

The umbilical cord biomaterial comprises of collagen (types I, III andIV; typically about 75%-80%% of the matrix of the biomaterial),fibronectin, elastin, and may further comprise glycosaminoglycans,(GAGs, e.g., hyaluronic acid) and/or proteoglycans. Typically, lamininis not present, or is present in trace amounts (i.e., less than 0.1% ofthe dry weight of the biomaterial). Typically, the umbilical cordbiomaterial comprises collagen types I, III, IV, V, VI and VII. Incertain embodiments, the umbilical cord biomaterial can comprisenon-structural components, such as, for example, one or more growthfactors, e.g., platelet-derived growth factors (PDGFs),vascular-endothelial growth factor (VEGF), fibroblast growth factor(FGF), transforming growth factor-β1, and the like. In certainembodiments, the umbilical cord biomaterial comprises growth factorssuch as FGF, b-FGF, EGF, IGF-1, PDGF and TGF-β. The composition of theumbilical cord biomaterial may thus be ideally suited to encourage themigration of fibroblasts and macrophages, and thus, e.g., the promotionof wound healing.

In one embodiment, the invention provides an umbilical cord biomaterialwherein at least 50% of the dry weight of the biomaterial is collagen I.In various more specific embodiments, at least 55%, 60%, 65% or 70% ofthe dry weight of the biomaterial is collagen I. In another specificembodiment, the invention provides an umbilical cord biomaterial whereinat most 5% of the dry weight of the biomaterial is collagen III. Invarious more specific embodiments, at most 4.9%, 4.8%, 4.7%, 4.6%, 4.5%,4.4%, 4.3%, 4.2%, 4.1%, 4.0%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%,3.2%, 3.1%, 3.0% or 2.9% of the dry weight of the biomaterial iscollagen III. In another specific embodiment, the invention provides anumbilical cord biomaterial wherein at least 4% of the dry weight of thebiomaterial is collagen IV. In various more specific embodiments, atleast 5%, 6%, 7%, 8%, 9%, 10% or 11% of the dry weight of saidbiomaterial is collagen IV. In another specific embodiment, theinvention provides an umbilical cord biomaterial wherein at most 4% ofthe dry weight of the biomaterial is elastin. In various more specificembodiments, at most 3.8%, 3.6%, 3.4%, 3.2%, 3.0%, 2.8%, 2.6%, 2.4%,2.2%, 2.0%, or 1.8% of the dry weight of the biomaterial is elastin. Inanother specific embodiment, at least 4% of the dry weight of thebiomaterial is glycosaminoglycan. In various more specific embodiments,at least 4.1%, 4.2%, 4.3%, 4.4%, 4.5% or 4.6% of the dry weight of saidbiomaterial is glycosaminoglycan.

The umbilical cord biomaterial may be used in a single-layered format,for example, as a single-layer sheet or an un-laminated membrane.Alternatively, the umbilical cord biomaterial may be used in adouble-layer or multiple-layer format, e.g., the umbilical cordbiomaterial may be laminated. Lamination can provide greater stiffnessand durability, for example, during the healing process. The umbilicalcord biomaterial may be, for example, laminated as described below (seeSection 5.1.7).

The umbilical cord biomaterial may further comprise collagen from anon-umbilical cord source. For example, one or more layers of umbilicalcord biomaterial may comprise, e.g., be coated or impregnated with, orlayered with, purified extracted collagen. Such collagen may beobtained, for example, from commercial sources, or may be producedaccording to known methods, such as those disclosed in U.S. Pat. Nos.4,420,339, 5,814,328, and 5,436,135, the disclosures of which are herebyincorporated by reference. Such collagen can also be obtained from aplacental source, including a placenta obtained from the same donor asthe umbilical cord biomaterial.

The umbilical cord biomaterial can comprise one or more compounds orsubstances that are not present in the umbilical cord material fromwhich the biomaterial is derived. Moreover, the umbilical cordbiomaterial can comprise non-naturally-occurring amounts of one or morecompounds or substances that are normally present in the umbilical cordfrom which the biomaterial is derived. For example, the umbilical cordbiomaterial can comprise, e.g., can be impregnated with, a bioactivecompound, such as those listed in Section 5.1.2, below. Such bioactivecompounds include, but are not limited to, small organic molecules(e.g., drugs), antibiotics (such as, for example, Clindamycin,Minocycline, Doxycycline, Gentamycin), hormones, growth factors,anti-tumor agents, anti-fungal agents, anti-viral agents, painmedications, anti-histamines, anti-inflammatory agents, anti-infectivesincluding but not limited to silver (such as silver salts, including butnot limited to silver nitrate and silver sulfadiazine), elementalsilver, antibiotics, bactericidal enzymes (such as lysozyme), woundhealing agents (such as cytokines including but not limited to PDGF,TGF; thymosin), hyaluronic acid as a wound healing agent, wound sealants(such as fibrin with or without thrombin), cellular attractant andscaffolding reagents (such as added fibronectin) and the like. In aspecific example, the umbilical cord biomaterial may be impregnated withat least one growth factor, for example, fibroblast growth factor,epithelial growth factor, etc. The biomaterial may also be impregnatedwith small organic molecules such as specific inhibitors of particularbiochemical processes e.g., membrane receptor inhibitors, kinaseinhibitors, growth inhibitors, anticancer drugs, antibiotics, etc.Impregnating the umbilical cord biomaterial with a bioactive compoundmay be accomplished, e.g., by immersing the biomaterial in a solution ofthe bioactive compound of the desired concentration for a timesufficient to allow the biomaterial to absorb and to equilibrate withthe solution. In a specific embodiment, the biomaterial so impregnatedis a dried biomaterial, and the solution partially or fully re-hydratesthe biomaterial, compared to an umbilical cord or umbilical cordmembrane in vivo. In another embodiment, the biomaterial is impregnatedprior to drying the biomaterial, e.g., to substantial dryness.

In other embodiments, the umbilical cord biomaterial may be combinedwith a hydrogel to form a composite. The use of any hydrogel compositionknown to one skilled in the art is encompassed within the invention,e.g., any of the hydrogel compositions disclosed in the followingreviews: Graham, 1998, Med. Device Technol. 9 (1): 18-22; Peppas et al.,2000, Eur. J. Pharm. Biopharm. 50 (1): 27-46; Nguyen et al., 2002,Biomaterials, 23 (22): 4307-14; Henincl et al., 2002, Adv. Drug Deliv.Rev 54 (1): 13-36; Skelhome et al., 2002, Med. Device. Technol. 13 (9):19-23; Schmedlen et al., 2002, Biomaterials 23: 4325-32. In a specificembodiment, the hydrogel composition is applied on the umbilical cordbiomaterial, that is, is disposed on the surface of the biomaterial. Thehydrogel composition for example, may be sprayed onto the umbilical cordbiomaterial or coated onto the surface of the biomaterial, or thebiomaterial may, for example, be soaked, bathed or saturated with thehydrogel composition. In another specific embodiment, the hydrogel issandwiched between two or more layers of umbilical cord biomaterial. Inan even more specific embodiment, the hydrogel is sandwiched between twolayers of umbilical cord biomaterial, wherein the edges of the twolayers of biomaterial are sealed so as to substantially or completelycontain the hydrogel.

The hydrogels useful in the methods and compositions of the inventioncan be made from any water-interactive, or water soluble polymer knownin the art, including but not limited to, polyvinylalcohol (PVA),polyhydroxyehthyl methacrylate, polyethylene glycol, polyvinylpyrrolidone, hyaluronic acid, alginate, collagen, gelatin, dextran orderivatives and analogs thereof.

In some embodiments, a composition comprises an umbilical cordbiomaterial of the invention, one or more bioactive compounds and ahydrogel. In other embodiments, a composition comprises an umbilicalcord biomaterial and a hydrogel composition that comprises one or morebioactive compounds. In yet another embodiment, a composition comprisesan umbilical cord biomaterial comprising one or more bioactive compoundsand a hydrogel composition comprising one or more bioactive compounds.The bioactive compounds can be, for example, one or more compounds asdescribed in Section 5.1.2, below.

5.1.2 Bioactive Compounds

The umbilical cord biomaterial of the invention can comprise (e.g., beimpregnated with or coated with) one or more bioactive or medicinalcompounds, such as small organic molecules (e.g., drugs), antibiotics,antiviral agents, antimicrobial agents, anti-inflammatory agents,antiproliferative agents, cytokines, enzyme or protein inhibitors,antihistamines, and the like. In various embodiments, the umbilical cordbiomaterial may be coated or impregnated with antibiotics (such asClindamycin, Minocycline, Doxycycline, Gentamycin), hormones, growthfactors, anti-tumor agents, anti-fungal agents, anti-viral agents, painmedications (including XYLOCAINE®, Lidocaine, Procaine, Novocaine,etc.), antihistamines (e.g., diphenhydramine, BENADRYL®, etc.),anti-inflammatory agents, anti-infectives including but not limited tosilver (such as silver salts, including but not limited to silvernitrate and silver sulfadiazine), elemental silver, antibiotics,bactericidal enzymes (such as lysozome), wound healing agents (such ascytokines including but not limited to PDGF (e.g., REGRANEX®), TGF;thymosin), hyaluronic acid as a wound healing agent, wound sealants(such as fibrin with or without thrombin), cellular attractant andscaffolding reagents (such as fibronectin), and the like, orcombinations of any of the foregoing, or of the foregoing and othercompounds not listed. Such impregnation or coating may be accomplishedby any means known in the art, and a portion or the whole of theumbilical cord biomaterial may be so coated or impregnated.

The umbilical cord biomaterial, or composites comprising umbilical cordbiomaterial, may comprise any of the compounds listed herein, withoutlimitation, individually or in any combination. Any of the biologicallyactive compounds listed herein may be formulated by known methods forimmediate release or extended release. Additionally, the umbilical cordbiomaterial may comprise two or more biologically active compounds indifferent manners; e.g., the biomaterial may be impregnated with onebiologically active compound and coated with another. In anotherembodiment, the umbilical cord biomaterial comprises one biologicallyactive compound formulated for extended release, and a secondbiologically active compound formulated for immediate release.

Wound healing requires adequate nutrition, particularly the presence ofiron, zinc, vitamin C, arginine, and the like. Thus, the umbilical cordbiomaterial may comprise, e.g., be impregnated or coated with, aphysiologically-available form of one or more nutrients required forwound healing. Preferably, the nutrient is formulated for extendedrelease.

The umbilical cord biomaterial, or composite comprising umbilical cordbiomaterial, may comprise an antibiotic. In certain embodiments, theantibiotic is a macrolide (e.g., tobramycin (TOBI®)), a cephalosporin(e.g., cephalexin (KEFLEX®)), cephradine (VELOSEF®)), cefuroxime(CEFTIN®, cefprozil (CEFZIL®), cefaclor (CECLOR®), cefixime (SUPRAX® orcefadroxil (DURICEF®), a clarithromycin (e.g., clarithromycin (Biaxin)),an erythromycin (e.g., erythromycin (EMYCIN®)), a penicillin (e.g.,penicillin V (V-CILLINK® or PEN VEEK®)) or a quinolone (e.g., ofloxacin(FLOXIN®), ciprofloxacin (CIPRO®) ornorfloxacin (NOROXIN®)),aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins,butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicolantibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, andthiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem andimipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, andcefpirome), cephamycins (e.g., cefbuperazone, cefinetazole, andcefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), macrolides (e.g., azithromycin, carbomycin, clarithomycin,dirithromycin, erythromycin, and erythromycin acistrate), amphomycin,bacitracin, capreomycin, colistin, enduracidin, enviomycin,tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, anddemeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans(e.g., furaltadone, and furazolium chloride), quinolones and analogsthereof (e.g., cinoxacin, ciprofloxacin, clinafloxacin, flumequine, andgrepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine,benzylsulfamide, noprylsulfamide, phthalylsulfacetamide,sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone,glucosulfone sodium, and solasulfone), cycloserine, mupirocin andtuberin.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, may comprise, e.g., be coated or impregnated with, anantifungal agent. Suitable antifungal agents include but are not limitedto amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal,flucytosine, miconazole, butoconazole, clotrimazole, nystatin,terconazole, tioconazole, ciclopirox, econazole, haloprogrin, naftifine,terbinafine, undecylenate, and griseofuldin.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, may comprise, e.g., be coated or impregnated with, ananti-inflammatory agent. Useful anti-inflammatory agents include, butare not limited to, non-steroidal anti-inflammatory drugs such assalicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal,salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin,sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin,ketorolac, dichlofenac, ibuprofen, naproxen, naproxen sodium,fenoprofen, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam,ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone,oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide;leukotriene antagonists including, but not limited to, zileuton,aurothioglucose, gold sodium thiomalate and auranofin; and otheranti-inflammatory agents including, but not limited to, methotrexate,colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, may comprise, e.g., be coated or impregnated with, anantiviral agent. Useful antiviral agents include, but are not limitedto, nucleoside analogs, such as zidovudine, acyclovir, gangcyclovir,vidarabine, idoxuridine, trifluridine, and ribavirin, as well asfoscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir,and the alpha-interferons.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, comprises, e.g., may be coated or impregnated with, acytokine receptor modulator. Examples of cytokine receptor modulatorsinclude, but are not limited to, soluble cytokine receptors (e.g., theextracellular domain of a TNF-α receptor or a fragment thereof, theextracellular domain of an IL-10 receptor or a fragment thereof, and theextracellular domain of an IL-6 receptor or a fragment thereof),cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β,interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokine receptorantibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptorantibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptorantibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptorantibodies, and anti-IL-12 receptor antibodies), anti-cytokineantibodies (e.g., anti-IFN antibodies, anti-TNF-α antibodies, anti-IL-10antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8(Abgenix)), and anti-IL-12 antibodies). In a specific embodiment, acytokine receptor modulator is IL-4, IL-10, or a fragment thereof. Inanother embodiment, a cytokine receptor modulator is an anti-IL-1antibody, anti-IL-6 antibody, anti-IL-12 receptor antibody, oranti-TNF-α antibody. In another embodiment, a cytokine receptormodulator is the extracellular domain of a TNF-α receptor or a fragmentthereof. In certain embodiments, a cytokine receptor modulator is not aTNF-α antagonist.

In a preferred embodiment, proteins, polypeptides or peptides (includingantibodies) that are utilized as immunomodulatory agents are derivedfrom the same species as the recipient of the proteins, polypeptides orpeptides so as to reduce the likelihood of an immune response to thoseproteins, polypeptides or peptides. In another preferred embodiment,when the subject is a human, the proteins, polypeptides, or peptidesthat are utilized as immunomodulatory agents are human or humanized.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, may also comprise, e.g., be coated or impregnated with. acytokine. Examples of cytokines include, but are not limited to, colonystimulating factor 1 (CSF-1), interleukin-2 (IL-2), interleukin-3(IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10(IL-10), interleukin-12 (IL-12), interleukin 15 (IL-15), interleukin 18(IL-18), insulin-like growth factor 1 (IGF-1), platelet derived growthfactor (PDGF), erythropoietin (Epo), epidermal growth factor (EGF),fibroblast growth factor (FGF) (basic or acidic), granulocyte macrophagestimulating factor (GM-CSF), granulocyte colony stimulating factor(G-CSF), heparin binding epidermal growth factor (HEGF), macrophagecolony stimulating factor (M-CSF), prolactin, and interferon (IFN),e.g., IFN-alpha, and IFN-gamma), transforming growth factor alpha(TGF-α), TGFβ1, TGFβ2, tumor necrosis factor alpha (TNF-α), vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF), etc.

The umbilical cord biomaterial may also comprise, e.g., be coated orimpregnated with, a hormone. Examples of hormones include, but are notlimited to, luteinizing hormone releasing hormone (LHRH), growth hormone(GH), growth hormone releasing hormone, ACTH, somatostatin,somatotropin, somatomedin, parathyroid hormone, hypothalamic releasingfactors, insulin, glucagon, enkephalins, vasopressin, calcitonin,heparin, low molecular weight heparins, heparinoids, synthetic andnatural opioids, insulin thyroid stimulating hormones, and endorphins.Examples of β-interferons include, but are not limited to, interferon β1-a and interferon β 1-b.

The umbilical cord biomaterial, or composite comprising umbilical cordbiomaterial, may also comprise, e.g., be coated or impregnated with, analkylating agent. Examples of alkylating agents include, but are notlimited to nitrogen mustards, ethylenimines, methylmelamines, alkylsulfonates, nitrosoureas, triazenes, mechlorethamine, cyclophosphamide,ifosfamide, melphalan, chlorambucil, hexamethylmelaine, thiotepa,busulfan, carmustine, streptozocin, dacarbazine and temozolomide.

The umbilical cord biomaterial, or a composite comprising umbilical cordbiomaterial, may also comprise, e.g., be coated or impregnated with, animmunomodulatory agent, including but not limited to methothrexate,leflunomide, cyclophosphamide, cyclosporine A, macrolide antibiotics(e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators, peptidemimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal,polyclonal, Fvs, ScFvs, Fab or F(ab)₂ fragments or epitope bindingfragments), nucleic acid molecules (e.g., antisense nucleic acidmolecules and triple helices), small molecules, organic compounds, andinorganic compounds. In particular, immunomodulatory agents include, butare not limited to, methothrexate, leflunomide, cyclophosphamide,cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics(e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids,steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), Tcell receptor modulators, and cytokine receptor modulators. Examples ofT cell receptor modulators include, but are not limited to, anti-T cellreceptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412(Boehringer), IDEC-CE9.Is (IDEC and SKB), mAb 4162W94, Orthoclone andOKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (ProductDesign Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies, anti-CD1 1aantibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g.,IDEC-114) (IDEC))) and CTLA4-immunoglobulin. In a specific embodiment, aT cell receptor modulator is a CD2 antagonist. In other embodiments, a Tcell receptor modulator is not a CD2 antagonist. In another specificembodiment, a T cell receptor modulator is a CD2 binding molecule,preferably MEDI-507. In other embodiments, a T cell receptor modulatoris not a CD2 binding molecule.

The umbilical cord biomaterial, or composite comprising umbilical cordbiomaterial, may also comprise, e.g., be coated or impregnated with aclass of immunomodulatory compounds known as IMIDS®. As used herein andunless otherwise indicated, the term “IMID®” and “IMIDS®” (CelgeneCorporation) encompasses small organic molecules that markedly inhibitTNF-α, LPS induced monocyte IL-1β and IL-12, and partially inhibit IL-6production. Specific immunomodulatory compounds are discussed below.

Specific examples of such immunomodulatory compounds, include, but arenot limited to, cyano and carboxy derivatives of substituted styrenessuch as those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl)isoindolines such asthose described in U.S. Pat. Nos. 5,874,448 and 5,955,476; the tetrasubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described inU.S. Pat. No. 5,798,368; 1-oxo and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines (e.g., 4-methylderivatives of thalidomide), including, but not limited to, thosedisclosed in U.S. Pat. Nos. 5,635,517, 6,476,052, 6,555,554, and6,403,613; 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or5-position of the indoline ring (e.g.,4-(4-amino-1,3-dioxoisoindoline-2-yl)-4-carbamoylbutanoic acid)described in U.S. Pat. No. 6,380,239; isoindoline-1-one andisoindoline-1,3-dione substituted in the 2-position with2,6-dioxo-3-hydroxypiperidin-5-yl (e.g.,2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one)described in U.S. Pat. No. 6,458,810; a class of non-polypeptide cyclicamides disclosed in U.S. Pat. Nos. 5,698,579 and 5,877,200;aminothalidomide, as well as analogs, hydrolysis products, metabolites,derivatives and precursors of aminothalidomide, and substituted2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles such as those described inU.S. Pat. Nos. 6,281,230 and 6,316,471; and isoindole-imide compoundssuch as those described in U.S. patent application Ser. No. 09/972,487filed on Oct. 5, 2001, U.S. patent application Ser. No. 10/032,286 filedon Dec. 21, 2001, and International Application No. PCT/US01/50401(International Publication No. WO 02/059106). The entireties of each ofthe patents and patent applications identified herein are incorporatedherein by reference. Immunomodulatory compounds do not includethalidomide.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo- and 1,3dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolinessubstituted with amino in the benzo ring as described in U.S. Pat. No.5,635,517 which is incorporated herein by reference. These compoundshave the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;-   1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;-   1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and-   1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.

Other specific immunomodulatory compounds belong to a class ofsubstituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as those described inU.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and 6,476,052, andInternational Patent Application No. PCT/US97/13375 (InternationalPublication No. WO 98/03502), each of which is incorporated herein byreference. Representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, or halo;

provided that R⁶ is other than hydrogen if X and Y are C═O and (i) eachof R¹, R², R³, and R⁴ is fluoro or (ii) one of R¹, R², R³, or R⁴ isamino.

Compounds representative of this class are of the formulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.,optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds belong to a class ofisoindole-imides disclosed in U.S. Patent Application Publication Nos.US 2003/0096841 and US 2003/0045552, and International Application No.PCT/US01/50401 (International Publication No. WO 02/059106), each ofwhich are incorporated herein by reference. Representative compounds areof formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R¹ is(C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,C(O)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, C(S)NHR³, or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H, (C₁-C₈)alkyl,benzyl, CH₂OCH₃, CH₂CH₂OCH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independently H,(C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl,aryl, halogen, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR⁵, (C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵, or adjacentoccurrences of R⁷ can be taken together to form a bicyclic alkyl or arylring.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Further examples of the compounds in this class include, but are notlimited to:[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide;(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamicacid tert-butyl ester;4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-acetamide;N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-carboxamide;2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide;3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide;{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methylacetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide;andN-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

Still other specific immunomodulatory compounds belong to a class ofisoindole-imides disclosed in U.S. Patent Application Publication Nos.US 2002/0045643, International Publication No. WO 98/54170, and U.S.Pat. No. 6,395,754, each of which is incorporated herein by reference.Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

Other representative compounds are of formula:

wherein:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X¹CH₂CH₂— in which X¹ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl.

Other representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

each of R¹, R², R³, and R⁴, independently of the others, is halo, alkylof 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one ofR¹, R², R³, and R⁴ is nitro or protected amino and the remaining of R¹,R², R³, and R⁴ are hydrogen; and

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Other representative compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

(i) each of R¹, R², R³, and R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, and R⁴ is —NHR⁵ and the remaining of R¹, R², R³, andR⁴ are hydrogen;

R⁵ is hydrogen, alkyl of 1 to 8 carbon atoms, or CO—R⁷—CH(R¹⁰)NR⁸R⁹ inwhich each of R⁷, R⁸, R⁹, and R¹⁰ is as herein defined; and

R⁶ is alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro.

Specific examples of the compounds are of formula:

in which:

one of X and Y is C═O and the other of X and Y is C═O or CH₂;

R⁶ is hydrogen, alkyl of 1 to 8 carbon atoms, benzyl, chloro, or fluoro;

R⁷ is m-phenylene, p-phenylene or —(C_(n)H_(2n))— in which n has a valueof 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X¹CH₂CH₂— in which X¹ is —O—,—S— or —NH—; and

R¹⁰ is hydrogen, alkyl of 1 to 8 carbon atoms, or phenyl.

Preferred immunomodulatory compounds are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione has thefollowing chemical structure:

The compound3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione has thefollowing chemical structure:

In another embodiment, specific immunomodulatory compounds encompasspolymorphic forms of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione such asForm A, B, C, D, E, F, G and H, disclosed in U.S. provisionalapplication no. 60/499,723 filed on Sep. 4, 2003, and U.S.non-provisional application Ser. No. 10/934,863, filed Sep. 3, 2004,both of which are incorporated herein by reference. For example, Form Aof 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is anunsolvated, crystalline material that can be obtained from non-aqueoussolvent systems. Form A has an X-ray powder diffraction patterncomprising significant peaks at approximately 8, 14.5, 16, 17.5, 20.5,24 and 26 degrees 2θ, and has a differential scanning calorimetrymelting temperature maximum of about 270° C. Form A is weakly or nothygroscopic and appears to be the most thermodynamically stableanhydrous polymorph of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidine-2,6-dionediscovered thus far.

Form B of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is ahemihydrated, crystalline material that can be obtained from varioussolvent systems, including, but not limited to, hexane, toluene, andwater. Form B has an X-ray powder diffraction pattern comprisingsignificant peaks at approximately 16, 18, 22 and 27 degrees 2θ, and hasendotherms from DSC curve of about 146 and 268° C., which are identifieddehydration and melting by hot stage microscopy experiments.Interconversion studies show that Form B converts to Form E in aqueoussolvent systems, and converts to other forms in acetone and otheranhydrous systems.

Form C of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is ahemisolvated crystalline material that can be obtained from solventssuch as, but not limited to, acetone. Form C has an X-ray powderdiffraction pattern comprising significant peaks at approximately 15.5and 25 degrees 2θ, and has a differential scanning calorimetry meltingtemperature maximum of about 269° C. Form C is not hygroscopic belowabout 85% RH, but can convert to Form B at higher relative humidities.

Form D of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is acrystalline, solvated polymorph prepared from a mixture of acetonitrileand water. Form D has an X-ray powder diffraction pattern comprisingsignificant peaks at approximately 27 and 28 degrees 2θ, and has adifferential scanning calorimetry melting temperature maximum of about270° C. Form D is either weakly or not hygroscopic, but will typicallyconvert to Form B when stressed at higher relative humidities.

Form E of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is adihydrated, crystalline material that can be obtained by slurrying3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in waterand by a slow evaporation of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione in asolvent system with a ratio of about 9:1 acetone:water. Form E has anX-ray powder diffraction pattern comprising significant peaks atapproximately 20, 24.5 and 29 degrees 2θ, and has a differentialscanning calorimetry melting temperature maximum of about 269° C. Form Ecan convert to Form C in an acetone solvent system and to Form G in aTHF solvent system. In aqueous solvent systems, Form E appears to be themost stable form. Desolvation experiments performed on Form E show thatupon heating at about 125° C. for about five minutes, Form E can convertto Form B. Upon heating at 175° C. for about five minutes, Form B canconvert to Form F.

Form F of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is anunsolvated, crystalline material that can be obtained from thedehydration of Form E. Form F has an X-ray powder diffraction patterncomprising significant peaks at approximately 19, 19.5 and 25 degrees2θ, and has a differential scanning calorimetry melting temperaturemaximum of about 269° C.

Form G of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is anunsolvated, crystalline material that can be obtained from slurryingforms B and E in a solvent such as, but not limited to, tetrahydrofuran(THF). Form G has an X-ray powder diffraction pattern comprisingsignificant peaks at approximately 21, 23 and 24.5 degrees 2θ, and has adifferential scanning calorimetry melting temperature maximum of about267° C.

Form H of3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)-piperidene-2,6-dione is apartially hydrated (about 0.25 moles) crystalline material that can beobtained by exposing Form E to 0% relative humidity. Form H has an X-raypowder diffraction pattern comprising significant peaks at approximately15, 26 and 31 degrees 2θ, and has a differential scanning calorimetrymelting temperature maximum of about 269° C.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl)isoindolines such asthose described in U.S. Pat. Nos. 5,874,448 and 5,955,476, each of whichis incorporated herein by reference. Representative compounds are offormula:

wherein Y is oxygen or H² and

each of R¹, R², R³, and R⁴, independently of the others, is hydrogen,halo, alkyl of 1 to 4 carbon atoms; alkoxy of 1 to 4 carbon atoms, oramino.

Other specific immunomodulatory compounds include, but are not limitedto, the tetra substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolinesdescribed in U.S. Pat. No. 5,798,368, which is incorporated herein byreference. Representative compounds are of formula:

wherein each of R¹, R², R³, and R⁴, independently of the others, ishalo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolinesdisclosed in U.S. Pat. No. 6,403,613, which is incorporated herein byreference. Representative compounds are of formula:

in which

Y is oxygen or H₂,

a first of R¹ and R² is halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, the second of R¹ and R², independently of thefirst, is hydrogen, halo, alkyl, alkoxy, alkylamino, dialkylamino,cyano, or carbamoyl, and

R³ is hydrogen, alkyl, or benzyl.

Specific examples of the compounds are of formula:

wherein a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms,alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl isof from 1 to 4 carbon atoms, cyano, or carbamoyl,

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl. Specificexamples include, but are not limited to,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

Other representative compounds are of formula:

wherein a first of R¹ and R² is halo, alkyl of from 1 to 4 carbon atoms,alkoxy of from 1 to 4 carbon atoms, dialkylamino in which each alkyl isof from 1 to 4 carbon atoms, cyano, or carbamoyl,

the second of R¹ and R², independently of the first, is hydrogen, halo,alkyl of from 1 to 4 carbon atoms, alkoxy of from 1 to 4 carbon atoms,alkylamino in which alkyl is of from 1 to 4 carbon atoms, dialkylaminoin which each alkyl is of from 1 to 4 carbon atoms, cyano, or carbamoyl,and

R³ is hydrogen, alkyl of from 1 to 4 carbon atoms, or benzyl.

Specific examples include, but are not limited to,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline.

Other specific immunomodulatory compounds include, but are not limitedto, 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-positionof the indoline ring described in U.S. Pat. No. 6,380,239 and co-pendingU.S. application Ser. No. 10/900,270, filed Jul. 28, 2004, which areincorporated herein by reference. Representative compounds are offormula:

in which the carbon atom designated C* constitutes a center of chirality(when n is not zero and R¹ is not the same as R²); one of X¹ and X² isamino, nitro, alkyl of one to six carbons, or NH—Z, and the other of X¹or X² is hydrogen; each of R¹ and R² independent of the other, ishydroxy or NH—Z; R³ is hydrogen, alkyl of one to six carbons, halo, orhaloalkyl; Z is hydrogen, aryl, alkyl of one to six carbons, formyl, oracyl of one to six carbons; and n has a value of 0, 1, or 2; providedthat if X¹ is amino, and n is 1 or 2, then R¹ and R² are not bothhydroxy; and the salts thereof.

Further representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH—Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH—Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2.

Specific examples include, but are not limited to,2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid and4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-cabamoyl-butyric acid,which have the following structures, respectively, and pharmaceuticallyacceptable salts, solvates, prodrugs, and stereoisomers thereof:

Other representative compounds are of formula:

in which the carbon atom designated C* constitutes a center of chiralitywhen n is not zero and R¹ is not R²; one of X¹ and X² is amino, nitro,alkyl of one to six carbons, or NH—Z, and the other of X¹ or X² ishydrogen; each of R¹ and R² independent of the other, is hydroxy orNH—Z; R³ is alkyl of one to six carbons, halo, or hydrogen; Z ishydrogen, aryl, or an alkyl or acyl of one to six carbons; and n has avalue of 0, 1, or 2; and the salts thereof.

Specific examples include, but are not limited to,4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyricacid, and2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pentanedioicacid, which have the following structures, respectively, andpharmaceutically acceptable salts, solvate, prodrugs, and stereoisomersthereof:

Other specific examples of the compounds are of formula:

wherein one of X¹ and X² is nitro, or NH—Z, and the other of X¹ or X² ishydrogen;

each of R¹ and R², independent of the other, is hydroxy or NH—Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons; and

n has a value of 0, 1, or 2;

provided that if one of X¹ and X² is nitro, and n is 1 or 2, then R¹ andR² are other than hydroxy; and if —COR² and —(CH₂)_(n)COR¹ aredifferent, the carbon atom designated C* constitutes a center ofchirality. Other representative compounds are of formula:

wherein one of X¹ and X² is alkyl of one to six carbons;

each of R¹ and R², independent of the other, is hydroxy or NH—Z;

R³ is alkyl of one to six carbons, halo, or hydrogen;

Z is hydrogen, phenyl, an acyl of one to six carbons, or an alkyl of oneto six carbons;

n has a value of 0, 1, or 2; and

if —COR² and —(CH₂)_(n)COR¹ are different, the carbon atom designated C*constitutes a center of chirality.

Still other specific immunomodulatory compounds include, but are notlimited to, isoindoline-1-one and isoindoline-1,3-dione substituted inthe 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl described in U.S.Pat. No. 6,458,810, which is incorporated herein by reference.Representative compounds are of formula:

wherein:

the carbon atoms designated * constitute centers of chirality;

X is —C(O)— or —CH₂—;

R¹ is alkyl of 1 to 8 carbon atoms or —NHR³;

R² is hydrogen, alkyl of 1 to 8 carbon atoms, or halogen; and

R³ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms,

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or —COR⁴ in which

R⁴ is hydrogen,

alkyl of 1 to 8 carbon atoms, unsubstituted or substituted with alkoxyof 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4 carbonatoms,

cycloalkyl of 3 to 18 carbon atoms,

phenyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms, or

benzyl, unsubstituted or substituted with alkyl of 1 to 8 carbon atoms,alkoxy of 1 to 8 carbon atoms, halo, amino, or alkylamino of 1 to 4carbon atoms.

The immunomodulatory compounds disclosed herein can either becommercially purchased or prepared according to the methods described inthe patents or patent publications disclosed herein. Further, opticallypure compounds can be asymmetrically synthesized or resolved using knownresolving agents or chiral columns as well as other standard syntheticorganic chemistry techniques.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein, and unless otherwise specified, the term “solvate” meansa compound of the present invention or a salt thereof, that furtherincludes a stoichiometric or non-stoichiometric amount of solvent boundby non-covalent intermolecular forces. Where the solvent is water, thesolvate is a hydrate.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of immunomodulatory compounds of the invention that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include derivatives of immunomodulatory compoundsof the invention that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can typically be prepared using well-known methods, such asthose described in 1 Burger's Medicinal Chemistry and Drug Discovery,172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design ofProdrugs (H. Bundgaard ed., Elselvier, New York 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide,” “biohydrolyzable ester,” “biohydrolyzablecarbamate,” “biohydrolyzable carbonate,” “biohydrolyzable ureide,”“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyl-oxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, amino acids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

As used herein, and unless otherwise specified, the term “stereoisomer”encompasses all enantiomerically/stereomerically pure andenantiomerically/stereomerically enriched compounds of this invention.

As used herein, and unless otherwise indicated, the term“stereomerically pure” or “enantiomerically pure” means that a compoundcomprises one stereoisomer and is substantially free of its counterstereoisomer or enantiomer. For example, a compound is stereomericallyor enantiomerically pure when the compound contains 80%, 90%, or 95% ormore of one stereoisomer and 20%, 10%, or 5% or less of the counterstereoisomer. In certain cases, a compound of the invention isconsidered optically active or stereomerically/enantiomerically pure(i.e., substantially the R-form or substantially the S-form) withrespect to a chiral center when the compound is about 80% ee(enantiomeric excess) or greater, preferably, equal to or greater than90% ee with respect to a particular chiral center, and more preferably95% ee with respect to a particular chiral center.

As used herein, and unless otherwise indicated, the term“stereomerically enriched” or “enantiomerically enriched” encompassesracemic mixtures as well as other mixtures of stereoisomers of compoundsof this invention (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40,65/35 and 70/30). Various immunomodulatory compounds of the inventioncontain one or more chiral centers, and can exist as racemic mixtures ofenantiomers or mixtures of diastereomers. This invention encompasses theuse of stereomerically pure forms of such compounds, as well as the useof mixtures of those forms. For example, mixtures comprising equal orunequal amounts of the enantiomers of a particular immunomodulatorycompounds of the invention may be used in methods and compositions ofthe invention. These isomers may be asymmetrically synthesized orresolved using standard techniques such as chiral columns or chiralresolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racematesand Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., etal., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of CarbonCompounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind., 1972).

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

The amount of the bioactive compound coating or impregnating theumbilical cord biomaterial may vary, and will preferably depend upon theparticular bioactive compound to be delivered, and the effect desired.For example, where the bioactive compound is an anti-inflammatory agent,the amount of the anti-inflammatory agent on or contained by theumbilical cord biomaterial is an amount sufficient to measurably reduceone or more symptoms or indicia of inflammation in a tissue contactedby, or proximal to, e.g., an umbilical cord biomaterial implant.

In various embodiments, the umbilical cord biomaterial of the inventionmay comprise, e.g., be coated or impregnated with, at least 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600,700, 800, 900, 100, 1250, 1500, 2000, 2500, 300, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000, 500000, 600000, 700000, 800000, 900000 or at least 1000000nanograms of a bioactive compound. In another embodiment, the umbilicalcord biomaterial of the invention may be coated with, or impregnatedwith, no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 300, 400, 500, 600, 700, 800, 900, 100, 1250, 1500, 2000, 2500,300, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000,900000 or at least 1000000 nanograms of a bioactive compound.

5.1.3 Conformation of the Umbilical Cord Biomaterial

The umbilical cord biomaterial may be formed into any shape orconformation that will facilitate its use in the methods of theinvention. For example, the umbilical cord biomaterial can be formedinto any shape or conformation that will facilitate, e.g., the occlusionof a tympanic membrane perforation, particularly in the context of atympanoplasty or myringoplasty; repair of a joint, ligament or tendon;etc. The umbilical cord biomaterial may, for example, be provided as anextended membrane, e.g., the entire membrane from a single umbilicalcord. The umbilical cord biomaterial can also be provided as square,rectangular, circular or oval shaped pieces, or may be cut to conformgenerally to the shape of, e.g., a tympanic membrane, tendon, or otherbodily structure. The umbilical cord biomaterial can be tubular. Invarious embodiments, umbilical cord biomaterial may be provided aspieces measuring approximately 1×1 cm, 1.5×1.5 cm, 2×2 cm, 2.5×2.5 cm,1×1.5 cm, 1×2 cm, 1×2.5 cm, 1×3 cm, 1×3.5 cm, 1×4 cm, 1×4.5 cm, 1×5 cm,1×5.5 cm, 1×6 cm, 1×6.5 cm, 1×7 cm, 1.5×2 cm, 1.5×2.5 cm, 1.5×3 cm,1.5×3.5 cm, 1.5×4 cm, 1.5×4.5 cm, 1.5×5 cm, 1.5×5.5 cm, 1.5×6 cm,1.5×6.5 cm, 1.5×7 cm, 2×2.5 cm, 2×3 cm, 2×3.5 cm, 2×4 cm, 2×4.5 cm, 2×5cm, 2×5.5 cm, 2×6 cm, 2×6.5 cm, 2×7 cm, 2.5×2.5 cm, 2.5×3 cm, 2.5×3.5cm, 2.5×4 cm, 2.5×4.5 cm, 2.5×5 cm, 2.5×5.5 cm, 2.5×6 cm, 2.5×6.5 cm,2.5×7 cm, 3×3 cm, 3×3.5 cm, 3×4 cm, 3×4.5 cm, 3×5 cm, 3×5.5 cm, 3×6 cm,3×6.5 cm, 3×7 cm, 3.5×3.5 cm, 3.5×4 cm, 3.5×4.5 cm, 3.5×5 cm, 3.5×5.5cm, 3.5×6 cm, 3.5×6.5 cm, 3.5×7 cm, 4×2.5 cm, 4×4 cm, 4×4.5 cm, 4×5 cm,4×5.5 cm, 4×6 cm, 4×6.5 cm, or 4×7 cm in size, or may be no smaller, orno larger, than 1×1 cm, 1.5×1.5 cm, 2×2 cm, 2.5×2.5 cm, 1×1.5 cm, 1×2cm, 1×2.5 cm, 1×3 cm, 1×3.5 cm, 1×4 cm, 1×4.5 cm, 1×5 cm, 1×5.5 cm, 1×6cm, 1×6.5 cm, 1×7 cm, 1.5×2 cm, 1.5×2.5 cm, 1.5×3 cm, 1.5×3.5 cm, 1.5×4cm, 1.5×4.5 cm, 1.5×5 cm, 1.5×5.5 cm, 1.5×6 cm, 1.5×6.5 cm, 1.5×7 cm,2×2.5 cm, 2×3 cm, 2×3.5 cm, 2×4 cm, 2×4.5 cm, 2×5 cm, 2×5.5 cm; 2×6 cm,2×6.5 cm, 2×7 cm, 2.5×2.5 cm, 2.5×3 cm, 2.5×3.5 cm, 2.5×4 cm, 2.5×4.5cm, 2.5×5 cm, 2.5×5.5 cm, 2.5×6 cm, 2.5×6.5 cm, 2.5×7 cm, 3×3 cm, 3×3.5cm, 3×4 cm, 3×4.5 cm, 3×5 cm, 3×5.5 cm, 3×6 cm, 3×6.5 cm, 3×7 cm,3.5×3.5 cm, 3.5×4 cm, 3.5×4.5 cm, 3.5×5 cm, 3.5×5.5 cm, 3.5×6 cm,3.5×6.5 cm, 3.5×7 cm, 4×2.5 cm, 4×4 cm, 4×4.5 cm, 4×5 cm, 4×5.5 cm, 4×6cm, 4×6.5 cm, or 4×7 cm, though the biomaterial may be cut to differentdimensions. In preferred embodiments, wherein the umbilical cordbiomaterial is used as a wound covering, the biomaterial is about 2.5cm×2.5 cm to 3.5 cm×3.5 cm, or, more preferably, about 3×3 cm². Longerand/or wider pieces can be formed by laminating two or more smallerpieces, as described elsewhere herein. Further, the biomaterial may beprovided as a sheet from which an end user may cut two or more pieces,or may be provided as a roll or strip.

The biomaterial may be provided to the end user either dry, orpre-wetted in a suitable physiologically-compatible, medically-usefulliquid, such as a saline solution. In one embodiment, the solutioncomprises one or more bioactive compounds, as described in Section5.1.2, above. Preferably, said bioactive compound is disposed onto orwithin the umbilical cord biomaterial such that the majority of thebioactive compound contacts the tympanic membrane at some point duringthe time the umbilical cord biomaterial contacts the tympanic membrane.

5.1.4 Methods of Making Umbilical Cord Biomaterial

The umbilical cord biomaterial of the invention can be made in a numberof ways. For example, the biomaterial is preferably produced by anymeans that preserves the biochemical and structural characteristics ofthe membrane's components—chiefly collagen, elastin, laminin, andfibronectin. That is, the biomaterial can be made so as to preserve, orsubstantially preserve, the native structure of the protein componentsof the biomaterial. The biomaterial may also be altered, e.g., theproteins of the biomaterial can be crosslinked, so as to improve thestrength (e.g., tensile strength) of the biomaterial. The biomaterialcan be completely, or substantially completely, decellularized prior touse, that is, can comprise only, or substantially only, an umbilicalcord outer membrane, or can be made to retain other components of theumbilical cord (e.g., Wharton's jelly, umbilical vessel(s), umbilicalcord cells, and the like). Generally, an umbilical cord is separatedfrom a placenta obtained by normal birth. The umbilical cord is thencleaned and disinfected, and optionally stored for further processing,e.g., decellularization and/or drying.

In one embodiment, the umbilical cord is separated from the placenta assoon as possible after delivery of the newborn. The umbilical cord maybe used immediately, or may be stored for 2-5 days from the time ofdelivery prior to any further treatment. Preferably, the expectantmother is screened prior to the time of birth, using standard techniquesknown to one skilled in the art, for communicable diseases including butnot limited to, HIV, HBV, HCV, HTLV, syphilis, CMV, and other viralpathogens known to contaminate umbilical cord tissue. One exemplarymethod for preparing the umbilical cord biomaterial of the inventioncomprises the following steps:

The umbilical cord is separated from the placental disc, and istypically massaged to remove umbilical cord blood. Optionally, theumbilical cord is sectioned into pieces of about 10 cm to about 15 cm inlength. The umbilical cord or umbilical cord sections can then be storedfor up to about 72 hours in a sterile, preferably buffered, salinesolution, such as 0.9% sterile NaCl solution. Preferably, the umbilicalcord is stored under refrigeration, at a temperature of about 1° C. toabout 5° C.

At this time, the umbilical cord can be slit or cut longitudinallyusing, e.g., a scalpel and forceps, grooved director, or the like. Thisallows the umbilical cord membrane to be laid flat, allowing, e.g.,removal of the Wharton's jelly, and/or one or more of the umbilical cordarteries, e.g., with a forceps. The umbilical cord membrane can also beprocessed further without cutting and opening the membrane. An umbilicalcord vessel, for example, can be removed from the cord by grasping thevessels with a forceps and gently pulling and massaging until the vesselis removed, leaving the umbilical cord membrane as an intact tube. In apreferred embodiment of deveining, the umbilical vein of a fresh (lessthan 48 hours after delivery) umbilical cord is canalized using theblunt probe of a vein stripper. The blunt probe is replaced with a smallbullet probe, and the vein is tied to the probe with thread. Thestripper is then removed, and the process is repeated with the umbilicalarteries.

The umbilical cord can be further processed “as is”, wherein the cordcomprises the umbilical cord membrane, vessels, and Wharton's jelly.

Continuing the embodiment, the umbilical cord biomaterial can besubstantially decellularized; that is, substantially all cellularmaterial and cellular debris (e.g., all visible cellular material andcellular debris) can removed from the biomaterial. Any decellularizingprocess known to one skilled in the art may be used, however, generallythe process used for decellularizing the umbilical cord biomaterial ofthe invention does not disrupt the native conformation of the proteinsmaking up the biomaterial. “Substantially decellularized,” as usedherein, means removal of at least 90% of the cells, more preferably atleast 95% of the cells, and most preferably at least 99% of the cellsassociated with the umbilical cord membrane. Decellularization can leavecellular material on the umbilical cord biomaterial; for example,decellularization can leave nuclear material detectable by4′,6-diamidino-2-phenylindole (DAPI) and still be considereddecellularized.

Decellularization can comprise physical scraping, for example, with asterile cell scraper, in combination with rinsing with a sterilesolution. The decellularization technique employed preferably does notresult in gross disruption of the anatomy of the umbilical cord membraneor alter the biomechanical properties of the umbilical cord membrane.

The decellularization of the umbilical cord biomaterial can comprisecontacting the membrane with a detergent-containing solution, such asone or more mild anionic or nonionic detergents, e.g., Triton X-100,sodium dodecyl sulfate, or the like, in an amount and for a timesufficient to decellularize the biomaterial. Any mild detergent, i.e., anon-caustic, low-foaming detergent, with a pH of about 6 to about 8, canbe used to decellularize the umbilical cord biomaterial. In a specificembodiment, the biomaterial is contacted with about 0.01-1% deoxycholicacid (e.g., deoxycholic acid sodium salt monohydrate) for about 30minutes to about 480 hours, preferably about 1 hour to about 240 hours,to decellularize the umbilical cord biomaterial. In a preferredembodiment, the umbilical cord biomaterial is decellularized in about 1%for about 20 days without scraping, followed by heat drying.

The biomaterial can be decellularized by any other method known to thosein the art, including freezing to form intracellular ice (including,e.g., vapor phase freezing). Where freezing is used to decellularize thebiomaterial, preferably a cryoprotectant is used, e.g.,polyvinylpyrollidone at, e.g., 10% w/v, or dialyzed hydroxyethyl starchat, e.g., 10% w/v, added to standard cryopreservation solutions such as,in a non-limiting example, DMEM comprising 10% DMSO and 10% fetal bovineserum.

Preferably, any native or exogenous protease activity is inhibited orprevented in the preparation of the biomaterial. Additives to thedecellularization, rinse and/or storage solutions such as metal ionchelators, for example 1,10-phenanthroline andethylenediaminetetraacetic acid (EDTA), create an environmentunfavorable to many proteolytic enzymes. Providing sub-optimalconditions for proteases (e.g., collagenase) assists in protectingumbilical cord biomaterial components such as collagen from degradationduring the cell lysis step. Suboptimal conditions for proteases may beachieved by formulating the decellularization solution to eliminate orlimit the amount of available calcium and zinc ions. Many proteases areactive in the presence of calcium and zinc ions and lose much of theiractivity in calcium and zinc ion free environments. Preferably, thedecellularization solution is prepared, in part, by selecting conditionsof pH, reduced availability of calcium and zinc ions, presence of metalion chelators and the use of proteolytic inhibitors specific forcollagenase, such that the solution will optimally lyse the nativeumbilical cord cells while protecting the umbilical cord biomaterialfrom proteolytic degradation. For example, a decellularization solutioncan include a buffered solution of water, pH 5.5 to 8, preferably pH 7to 8, free from calcium and zinc ions and including a metal ion chelatorsuch as EDTA. Decellularization can take place at, e.g., between 0° C.and 25° C., preferably below about 10° C., to reduce protease activity.

It is preferred that the decellularization treatment also limits thegeneration of new immunological sites. Since enzymatic degradation of,e.g., collagen is believed to lead to heightened immunogenicity, theinvention encompasses treatment of the umbilical cord biomaterial withenzymes, e.g., nucleases, that are effective in inhibiting cellularmetabolism, protein production and cell division, that minimizeproteolysis of the components of the umbilical cord biomaterial thuspreserving the underlying architecture of the amniotic biomaterial.Examples of nucleases that can be used in accordance with the methods ofthe invention are those effective in digestion of native cell DNA andRNA including both exonucleases and endonucleases. A non-limitingexample of nucleases that can be used in accordance with the methods ofthe invention include exonucleases that inhibit cellular activity, e.g.,DNase I (SIGMA Chemical Company, St. Louis, Mo.) and RNase A (SIGMAChemical Company, St. Louis, Mo.) and endonucleases that inhibitcellular activity, e.g., EcoRI (SIGMA Chemical Company, St. Louis, Mo.)and HindIII (SIGMA Chemical Company, St. Louis, Mo.). It is preferablethat the selected nucleases are applied in a physiological buffersolution which contains ions, e.g., magnesium, calcium, which areoptimal for the activity of the nuclease. Preferably, the ionicconcentration of the buffered solution, the treatment temperature andthe length of treatment are selected by one skilled in the art byroutine experimentation to assure the desired level of nucleaseactivity. The buffer is preferably hypotonic to promote access of thenucleases to cell interiors.

In another embodiment of the invention, the umbilical cord biomaterialis not decellularized prior to drying.

In another embodiment of the above steps, the umbilical cord, afterinitial processing, is briefly rinsed in saline to remove blood from theumbilical cord surface. The umbilical cord is then immersed in a colddeoxycholic acid solution at a concentration of about 0.1% to about 10%,and, in a specific embodiment, about 0.1% to about 2.0%. The umbilicalcord is then incubated in this solution at between about 1° C. to about8° C. for about 5 days to about 6 months. In specific embodiments, theumbilical cord is immersed, for example, for about 5 to about 15 days;about 5 to about 30 days, about 5 to about 60 days, or for up to aboutone year. Typically, the deoxycholic acid solution is replaced duringincubation every 2-5 days. In another specific embodiment, the umbilicalcord is immersed in a deoxycholic acid solution at a concentration ofabout 1% at a temperature of 0° C. to about 8° C. for about 5 days toabout 15 days. This incubation serves two purposes. First, it allowstime for serological tests to be performed on the umbilical cord and/orumbilical cord blood, so that umbilical cords failing to meetserological criteria are not processed further. Second, the longerincubation improves the removal of epithelial cells and fibroblasts,which allows for a significant reduction in the amount of time spentdecellularizing the umbilical cord membrane by physically scraping. Theumbilical cord biomaterial can then be dried as described below.

Following decellularization, the umbilical cord biomaterial is generallywashed to assure removal of cellular debris (e.g., cellular proteins,cellular lipids, cellular nucleic acids, extracellular debris such asextracellular soluble proteins, lipids and proteoglycans, and the like).The wash solution can be de-ionized water or an aqueous hypotonicbuffer. Preferably, the umbilical cord biomaterial is gently agitated,e.g., for 15-120 minutes in the detergent, e.g., on a rocking platform,to assist in the decellularization. The umbilical cord biomaterial,after detergent decellularization, can again be physicallydecellularized as described above; the physical and detergentdecellularization steps may be repeated as necessary, as long as theintegrity of the umbilical cord biomaterial is maintained, until novisible'cellular material and cellular debris remain.

In certain embodiments, the umbilical cord biomaterial is driedimmediately (i.e., within 30 minutes) after decellularization and/orwashing. Alternatively, when further processing is not done immediately,the umbilical cord biomaterial may be refrigerated, e.g., stored at atemperature of about 1° C. to about 20° C., preferably from about 2° C.to about 8° C., for up to 28 days prior to drying. When the umbilicalcord biomaterial, e.g., decellularized umbilical cord biomaterial, isstored for more than three days, the sterile solution covering theumbilical cord biomaterial is preferably changed periodically, e.g.,every 1-3 days.

In certain embodiments, when the umbilical cord biomaterial is notrefrigerated after washing, the biomaterial can be washed, e.g., washedat least 3 times, prior to proceeding to the next step of thepreparation. In other embodiments, when the umbilical cord biomaterialhas been refrigerated and the sterile solution has been changed once,the umbilical cord biomaterial can be washed at least twice prior to thenext step of the preparation. In yet other embodiments, when theumbilical cord biomaterial has been refrigerated and the sterilesolution has been changed twice or more, the umbilical cord biomaterialcan be washed at least once prior to proceeding to the next step.

The final step in this embodiment comprises drying the decellularizedumbilical cord membrane to produce the umbilical cord biomaterial of theinvention. Any method of drying the umbilical cord membrane can be used.For example, the membrane can be dried using heat, one or morehygroscopic compounds, freeze-drying, vacuum, microwaving, simpleevaporation, and the like, or combinations of these methods. Preferably,the biomaterial is dried under vacuum.

The umbilical cord biomaterial can be dried in any useful conformation.Preferably, the umbilical cord biomaterial is dried so as to produce aflat, dry sheet. The biomaterial can also be dried as a tube, strip,spiral, string or rope, or the like. For three-dimensional shapes, thebiomaterial can be placed onto, or into, a form and dried, so that thedried biomaterial assumes the shape of the form or a part thereof. In aspecific embodiment, for example, the umbilical cord membrane can besupported by rubber hose or tubing inserted from one end, andfreeze-dried to form a dried tube of the umbilical cord biomaterial. Atthis point, the umbilical cord biomaterial can be, e.g., part of acomplete umbilical cord that has been washed and rinsed; an umbilicalcord biomaterial comprising Wharton's jelly but lacking vessels, anumbilical cord biomaterial that has had the interior components of theumbilical cord removed, and has been decellularized, etc. In each case,the biomaterial can be dried.

In a specific embodiment, an exemplary method for drying the umbilicalcord biomaterial comprises the following steps:

Assembly of the umbilical cord biomaterial for drying. The umbilicalcord biomaterial is removed from the sterile solution, and the excessfluid is gently squeezed out. The umbilical cord biomaterial is thengently stretched until it is flat with the epithelial side facing in adownward position, e.g., on a tray. The umbilical cord biomaterial isthen placed on a drying frame, preferably a plastic mesh drying frame(e.g., QUICK COUNT® Plastic Canvas, Uniek, Inc., Waunakee, Wis.). Inother embodiments, the drying frame may be any autoclavable material,including but not limited to a stainless steel mesh. Once the umbilicalcord biomaterial is positioned on the drying frame, a sterile gauze canbe placed on the drying platform of a heat dryer (or gel-dryer) (e.g.,Model 583, Bio-Rad Laboratories, Hercules, Calif.), so that an areaslightly larger than the umbilical cord biomaterial resting on theplastic mesh drying frame is covered. Preferably, the total thickness ofthe gauze layer does not exceed the thickness of one folded 4×4 gauze.Any heat drying apparatus may be used that is suitable for dryingsheet-like material. The drying frame is placed on top of the gauze onthe drying platform so that the edges of the plastic frame extend abovebeyond the gauze edges, preferably between 0.1-1.0 cm, more preferably0.5-1.0 cm. In some embodiments, another plastic framing mesh is placedon top of the umbilical cord biomaterial. In another embodiments, asheet of thin plastic (e.g., SW 182, clear PVC, AEP Industries Inc.,South Hackensack, N.J.) or a biocompatible silicone is placed on top ofthe biomaterial covered mesh so that the sheet extends well beyond allof the edges. In this embodiment, the second mesh frame is not needed.

In an alternative embodiment, the umbilical cord biomaterial is placedone or more sterile sheets of TYVEK® material (e.g., a sheet of TYVEK®for medical packaging, DuPont TYVEK®, Wilmington, Del.), optionally,with one sheet of TYVEK® on top of the biomaterial (prior to placing theplastic film). This alternate process will produce a smoother version ofthe biomaterial (i.e., without the pattern of differential fibercompression regions along and perpendicular to the axis of thematerial), which may be advantageous for certain applications, such asfor example for use as a matrix for expansion of cells.

Drying the umbilical cord biomaterial. In a preferred embodiment, theinvention encompasses heat drying the umbilical cord biomaterial of theinvention under vacuum. While the drying under vacuum may beaccomplished at any temperature from about 0° C. to about 60° C., theumbilical cord biomaterial is preferably dried at between about 35° C.and about 50° C., and most preferably at about 50° C. It should be notedthat some degradation of the collagen is to be expected at temperaturesabove 50° C. The drying temperature is preferably set and verified usinga calibrated digital thermometer using an extended probe. Any amount ofvacuum that can be conveniently generated can be used, but preferably,the vacuum pressure is set to about −22 inches of Hg. The drying step iscontinued until the umbilical cord biomaterial is substantially dry,that is, contains less than 20% water by weight, and preferably, about3-12% water by weight as determined for example by a moisture analyzer.To accomplish this, the umbilical cord biomaterial may be heat-vacuumdried, e.g., for approximately 60 minutes to achieve a dehydratedumbilical cord biomaterial. In some embodiments, the umbilical cordbiomaterial is dried for about 30 minutes to 2 hours, preferably about60 minutes. Although not intending to be bound by any mechanism ofaction, it is believed that low (e.g., <50° C.) heat coupled with vacuumpressure allows the umbilical cord biomaterial to achieve the dehydratedstate without denaturing collagen in the biomaterial. After completionof the drying process in accordance with the invention, the umbilicalcord biomaterial can be cooled down, e.g., for approximately twominutes, with the vacuum pump running.

Packaging and Storing of the Umbilical Cord Biomaterial. Once theumbilical cord biomaterial is dried, the biomaterial is gently liftedoff the drying frame. Preferably, handling of the umbilical cordbiomaterial at this stage is done with sterile gloves. The umbilicalcord biomaterial can be placed in a sterile container, e.g., peel pouch.When dried, the umbilical cord biomaterial produced in accordance withthe methods of the invention may be stored at room temperature for anextended period of time as described supra.

In another embodiment, the umbilical cord biomaterial is prepared asabove, but is not decellularized. That is, the umbilical cord membraneis obtained and dried, but the cells associated with the umbilical cordmembrane are not removed. The final, dried product thus comprises, e.g.,the umbilical cord membrane and/or umbilical cord vessel(s), as well ascellular components.

The umbilical cord biomaterial can be dehydrated by other methods inplace of, or in addition to, the vacuum-drying method outlined above.For example, in one embodiment, the biomaterial can be freeze dried.Typically, umbilical cord biomaterial can be frozen at a temperaturebetween about −170° C. and about 0° C. for time sufficient for thebiomaterial to completely freeze. The frozen biomaterial is freeze-driedprocess during which the ice crystals will be removed or avoided bysublimation under vacuum.

In another embodiment, the biomaterial can be dehydrated using asolvent. For example, umbilical cord biomaterial can be dehydratedusing, e.g., ethanol and acetone. In this specific embodiment, thebiomaterial can be, e.g., soaked for a time in a series ofethanol-acetone mixtures (e.g., 20%, 40%, 60%, 80% and 100% ethanol, ora similar progression of equivalent solvents that act to extract water)such that the water inside the biomaterial is gradually replaced by theorganic solvent. After the final soak, the biomaterial can be placed ina well-ventilated place at room temperature (about 23° C.) for a timesufficient for the solvent to evaporate. The biomaterial can alternatelybe vacuum-dried after the solvent soak.

In another embodiment, the biomaterial can also be dehydrated by freezedrying. For example, in a specific embodiment in a combination of theabove two processes, the processed membrane can be first frozen and thentransferred to a water miscible organic solvent. Ice crystals inside themembrane tissue may then be dissolved and replaced by the organicsolvent using a series of progressive solvent soaks as described above.After the final soak, the biomaterial can be placed in a well-ventilatedplace at room temperature (e.g., about 20° C. to about 25° C.) for atime sufficient for the ethanol to evaporate. The biomaterial canalternately be vacuum-dried after the 100% ethanol soak.

Non-heat drying processes may be preferred if the porous structure ofthe biomaterial and/or bioactivity of the biological substances withinthe umbilical cord membrane need to be preserved, for example, if thebiomaterial is to be used as a substrate or matrix for the transport ofstem cells to a graft site, or if, e.g., the biomaterial is to bepreloaded with a heat-sensitive drug as a drug release device, or if,e.g., the biomaterial is preloaded with a heat sensitive drug as a drugrelease device.

When the above steps are complete, the membrane generally primarilycomprises collagen (types I, III, IV, V, VI and VII), glycosaminoglycans(particularly hyaluronic acid); and growth factors, particularlyfibroblast growth factor (FGF), basic fibroblast growth factor (b-FGF),epidermal growth factor (EGF), insulin-like growth factor I (IGF-I),platelet-derived growth factor (PDGF) and transforming growth factorbeta (TGF-β).

5.1.5 Storage and Handling of Umbilical Cord Biomaterial

Dehydrated umbilical cord biomaterial may be stored, e.g., as dehydratedsheets, at room temperature (e.g., 25° C.) prior to use. In certainembodiments, the umbilical cord biomaterial can be stored at atemperature of at least 10° C., at least 15° C., at least 20° C., atleast 25° C., or at least 29° C. Preferably, umbilical cord biomaterial,in dehydrated form, is not refrigerated. In some embodiments, theumbilical cord biomaterial may be refrigerated at a temperature of about2° C. to about 8° C. The umbilical cord biomaterial produced accordingto the methods of the invention can be stored at any of the specifiedtemperatures for 12 months or more with no alteration in biochemical orstructural integrity (e.g., no degradation), without any alteration ofthe biochemical or biophysical properties of the umbilical cordbiomaterial. The biomaterial can be stored for several years with noalteration in biochemical or structural integrity (e.g., nodegradation), without any alteration of the biochemical or biophysicalproperties of the biomaterial. The biomaterial can be stored in anycontainer suitable for long-term storage. Preferably, the umbilical cordbiomaterial of the invention is stored in a sterile double peel-pouchpackage.

The umbilical cord biomaterial, in embodiments in which the material hasbeen dried, may be hydrated prior to use, using, e.g., a sterilephysiological buffer. In a specific embodiment, the sterile salinesolution is a 0.9% NaCl solution. In some embodiments the sterile salinesolution is buffered. In certain embodiments, the hydration of theumbilical cord biomaterial requires at least 2 minutes, at least 5minutes, at least 10 minutes, at least 15 minutes, or at least 20minutes. In a preferred embodiment, the hydration of the umbilical cordbiomaterial is complete within 5 minutes. In yet another preferredembodiment, the hydration of the umbilical cord biomaterial of theinvention is complete within 10 minutes. In yet another embodiment, thehydration of the umbilical cord biomaterial takes no more than 10minutes. Once hydrated, the umbilical cord biomaterial can be maintainedin solution, e.g., in sterile 0.9% NaCl solution, for up to six months,with a change of solution, e.g., every three days.

5.1.6 Sterilization

Sterilization of the umbilical cord biomaterial may be accomplished byany medically-appropriate means, preferably means that do notsignificantly alter the tertiary and quaternary structure of thebiomaterial proteins. Sterilization can be accomplished, for example,using gas, e.g., ethylene dioxide. Sterilization can be accomplishedusing radiation, for example, gamma radiation, and is preferably done byelectron beam irradiation using methods known to one skilled in the art,e.g., Gorham, D. Byrom (ed.), 1991, Biomaterials, Stockton Press, NewYork, 55-122. Any dose of radiation sufficient to kill at least 99.9% ofbacteria or other potentially contaminating organisms is within thescope of the invention. In a preferred embodiment, a dose of at least18-25 kGy is used to achieve the terminal sterilization of thebiomaterial.

5.1.7 Laminates

The umbilical cord biomaterial may be laminated to provide greaterload-bearing capacity and durability during the healing process.Laminates of the umbilical cord biomaterial can comprise biomaterialfrom a single umbilical cord, wherein the composition is folded once, ora plurality of times, longitudinally or laterally, or both. Laminates ofthe biomaterial can also comprise two or more sheets of biomaterial.

The umbilical cord biomaterial, being anisotropic, has two orientations,longitudinal (that is, along the length of the umbilical cord membrane)and lateral (that is, around the width of the umbilical cord). Where alaminate comprises two sheets of the umbilical cord biomaterial, thesheets can be laminated so that each sheet is oriented the same way(e.g., each sheet oriented longitudinally), or such that at least onesheet is oriented laterally and one longitudinally. In otherembodiments, each of the layers of umbilical cord biomaterial can belaminated in any orientation with respect to any other layer of thebiomaterial in the laminate.

The umbilical cord has a sidedness; that is, the umbilical cordbiomaterial has an epithelial side (that is, the side towards theinterior of the umbilical cord) and a mesothelial side (that is, theside towards the exterior of the umbilical cord). Laminates can comprisetwo or more layers of the umbilical cord biomaterial in any sidednessconfiguration. For example, laminates can comprise layers of umbilicalcord biomaterial in which only the endothelial sides of the layers arein contact; only the mesothelial sides of the layers are intact; or acombination of both. In one embodiment, a laminate comprises fourlayers, wherein two sets of two layers, contacted endothelial tomesothelial sides, are contacted by the exposed mesothelial side suchthat the two faces of the laminate show the endothelial sides.

Umbilical cord biomaterial can be laminated, e.g., by folding a singlesheet of biomaterial, or by stacking 2 or more layers of the biomaterialone atop the other, and sealing or drying. The biomaterial may belaminated either dry or after rehydration. Alternatively, two or morelayers of, e.g., umbilical cord biomaterial, or composition comprisingan umbilical cord biomaterial, can be laminated prior to initial dryingafter cell removal, e.g., after a cell scraping step (see Examples,below). If laminated prior to the initial drying, 2 or more biomateriallayers can be stacked one atop the other and subsequently dried, using,for example, a freeze-drying process, or drying under moderate heat withor without vacuum. The heat applied preferably is not so intense as tocause breakdown or decomposition of the protein components, especiallythe collagen, of the umbilical cord biomaterial. Typically, the heatapplied is less than about 70° C., preferably less than about 60° C.,and, more preferably, is approximately 50° C. Lamination time varieswith, e.g., the number of layers being laminated, but typically takes1-2 hours at 50° C. Thus, a method of preparing a laminate using acomposition comprising umbilical cord membrane comprises layering aplurality of said membranes in contact with each other to form alaminate. In some embodiments, each of said membranes comprises lessthan 20% water by weight prior to said layering. In certain embodiments,said laminate is dried to less than 20% water by weight after saidlayering.

The biomaterial may also be laminated using an adhesive applied between2 or more layers of biomaterial or umbilical cord biomaterial orcomposition comprising an umbilical cord biomaterial. Such an adhesiveis preferably appropriate for medical applications, and can comprise anatural biological adhesive, for example fibrin glue, a syntheticadhesive, or combinations thereof. The adhesive may further bechemically converted from precursors during the lamination process.

Laminates of the umbilical cord biomaterial can comprise, for example,umbilical cord biomaterial that has been decellularized, biomaterialthat retains the cellular material (that is, where the cells have beenkilled, but not removed), or biomaterial comprising living umbilicalcord cells or cells of another type (e.g., where cells have beencultured on a sheet of umbilical cord biomaterial).

A laminate can comprise a second type of material, e.g., one or morelayers of umbilical cord biomaterial can be layered with one or morelayers of a second biologically-compatible material, e.g., a sheetlikematerial such as, e.g., amniotic membrane. Where the second material hasa “grain” or orientation, the umbilical cord biomaterial can belaminated such that the biomaterial lies with its longitudinal directionalong, or alternatively across, the grain of the second material. In aspecific, preferred embodiment, the umbilical cord membrane biomaterialis laminated with at least one other layer of a second material that hasa relatively high load-bearing capacity. The umbilical cord biomaterialcan also be laminated with a non-biological material, e.g., plastic,e.g., TYVEK® or the like. In a preferred embodiment, the umbilical cordbiomaterial laminate comprises two layers of the biomaterial and onelayer of plastic, e.g., TYVEK®, such that the plastic is sandwichedbetween the two layers of umbilical cord biomaterial, and theendothelial sides of the biomaterial contact the plastic. In anotherpreferred embodiment, one layer of umbilical cord membrane is placed ona plastic sheet with the epithelial side down, and a second piece ofumbilical cord membrane is placed on the first epithelial side up. Theresulting product is then heat-dried to produce an umbilical cordbiomaterial laminate.

In various embodiments, the second material has a load-bearing capacityor tensile strength, for, e.g., a 2-centimeter wide section, of at least25 milliPascals (mPa), 50 mPa, 75 mPa, 100 mPa, 125 mPa, 150 mPa, 175mPa, 200 mPa, 225 mPa, 250 mPa, 275 mPa, 300 mPa, 325 mPa, 350 mPa, 375mPa, 400 mPa, 425 mPa, 450 mPa, 475 mPa, 500 mPa, 750 mPa, 1000 mPa,1250 mPa, 1500 mPa, 1750 mPa or 2000 mPa. Load-bearing capacities ofsections that are wider or narrower would be accordingly more or less.Such a second material can be sheetlike, or can be formed into a shapesuitable for a particular application, and the umbilical cordbiomaterial molded to the shape of the second material. For example, thesecond material can be, e.g., a material suitable for, and shaped for,tendon or ligament repair. The umbilical cord biomaterial can be wrappedaround such a second material so that the exterior of the laminate isbiologically compatible, and the interior is load-bearing.

The load-bearing capacity of a particular umbilical cord biomateriallaminate, or piece of umbilical cord biomaterial, can be tested bystandard methods known in the art, such as ASTM D1708 (Standard TestMethod for Tensile Properties of Plastics).

In one embodiment, the laminate comprises at least two sheets ofumbilical cord biomaterial approximately the same size and shape laidone atop the other so that the shape is substantially maintained. Such alaminate can be trimmed to finalize a particular shape. In anotherembodiment, the laminate comprises two or more sheets of umbilical cordbiomaterial, wherein a portion of each of the sheets overlaps another.In this embodiment, several overlapping sheets of umbilical cordbiomaterial can be laminated to form a larger sheet of the biomaterialthan would be possible from a single umbilical cord. In a specificembodiment, such a laminate of overlapping sheets of the biomaterial canitself be laminated with another layer of a material, e.g., anotheroverlapping biomaterial laminate; individual sheets of umbilical cordbiomaterial; another type of biomaterial, e.g., an amnioticmembrane-derived biomaterial; an artificial sheet or film; etc.

5.1.8 Stem Cells

The umbilical cord biomaterial as described herein can also comprisestem or progenitor cells. The umbilical cord biomaterial can comprise,e.g., mesenchymal or mesenchymal-like stem cells, for example, thosedescribed in U.S. Pat. Nos. 5,486,359, 6,261,549 and 6,387,367, orplacental stem cells such as those described in U.S. ApplicationPublication Nos. 2002/0123141, 2003/0032179 and 2003/0180269. However,the umbilical cord biomaterial may comprise stem or progenitor cells,preferably mammalian stem or progenitor cells, from any tissue source.The umbilical cord biomaterial can comprise embryonic stem cells orembryonic germ cells.

The umbilical cord biomaterial and stem or progenitor cells can becombined, e.g., in advance of a procedure in which the biomaterial iscontacted with an individual having a disease, disorder or conditionthat would be amenable to treatment using an umbilical cord biomaterial.For example, stem cells can be contacted with, e.g., disposed onto, thebiomaterial sufficiently in advance of such a procedure for a plurality,a majority, or substantially all of the stem cells to adhere to thebiomaterial. The stem cells can be contacted with the biomaterialimmediately before the biomaterial is contacted with the individual. Thestem cells can also be contacted with the biomaterial in situ, after thebiomaterial is contacted with the individual. The number of stem orprogenitor cells disposed onto the surface of the umbilical cordbiomaterial may vary, but may be at least about 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹, or1×10¹²; or may be no more than 1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹, or 1×10¹² stem orprogenitor cells.

The stem cells, at any of the times noted above, can be contacted withone or more differentiation-modulating agents, for example, thedifferentiation-modulating agents described in U.S. ApplicationPublication Nos. 2003/0235909 and/or 2004/0028660, the disclosures ofwhich are incorporated by reverence in their entireties herein, orInternational Application Publication No. WO 03/087333. Methods ofdifferentiating stem cells to, for example, epidermal, mesodermal, andother cell types are known in the art, and are described, e.g., in U.S.Application Publication No. 2004/0028660.

5.2 Uses of Umbilical Cord Biomaterial

The umbilical cord biomaterial can be used in a variety of medicalapplications. The uses outlined in this section are non-limitingexamples of such applications.

5.2.1 Repair of Joints, Ligaments, and Tendons

The umbilical cord biomaterial of the invention can be used, forexample, to repair or replace ligaments, tendons and/or cartilage. Thebiomaterial can be contacted with a ligament, tendon or cartilage in anymedically-acceptable manner that tends to facilitate healing of a defectin the ligament, tendon or cartilage.

The umbilical cord biomaterial can be used, for example, to repair adefect in a tendon, ligament or cartilage where the defect is a tear,that is, a defect that is less than a complete failure. The repaircomprises contacting the tendon, ligament or cartilage with theumbilical cord biomaterial such that a part, or preferably all, of thedefect is covered by the biomaterial. The umbilical cord biomaterial canbe, for example, wrapped around a tendon or ligament at the site of thedefect, or a patch of umbilical cord biomaterial of sufficient size tocover the defect can be placed on the defect. The biomaterial can beheld in place using, e.g., a biologically-acceptable glue, e.g., atissue glue, or can be sutured in place. Preferably, the umbilical cordbiomaterial is held in place such that the biomaterial assumes at leastpart of the biomechanical load normally associated with the functionaltendon, ligament or cartilage.

The umbilical cord biomaterial can be used, for example, to repair adefect in a tendon, ligament or cartilage where the defect is a completerupture or failure of a tendon, ligament or cartilage. The repaircomprises contacting the tendon, ligament or cartilage with theumbilical cord biomaterial so as to partially, or preferably completely,cover the defect. Preferably, the contacting is such that thebiomaterial assumes some, or all of the biomechanical load normallyassumed by the tendon, ligament or cartilage. For example, a rupture ina tendon can be repaired by contacting the tendon at the site of therupture with the biomaterial and affixing the biomaterial in place. Thetwo parts of the tendon at the site of the rupture are preferablybrought into contact with each other, and the biomaterial is preferablywrapped around the site of the break. The biomaterial can be sutured tothe two parts of the ruptured tendon such that it acts as a splint,holding the to ends of the rupture together. The biomaterial can bewrapped around such a rupture once, or preferably a plurality of times.

The umbilical cord biomaterial can be used, for example, to replace atendon or ligament, or to support a joint where one or more of thenative ligaments is weakened. In such an embodiment, the biomaterialattaches two bones, or a muscle and bone, in place of a damaged,diseased or ruptured tendon or ligament, e.g., an anterior cruciateligament.

In a specific embodiment, for example, to replace a cruciate ligament,e.g., an anterior cruciate ligament, tunnels can be drilled onto thefemoral and tibial heads adjoining the knee. The biomaterial, e.g.,folded lengthwise a plurality of times into a rope-like conformation,can be drawn through the tunnels, and the ends fastened to therespective bones. Such fastening can be accomplished by any means knownin the art, e.g., using screws, staples, or the like. In a specificembodiment, the ends of the umbilical cord biomaterial are folded overto provide a portion of the biomaterial of greater thickness forfastening. Such replacement can comprise removing the native ligamentduring replacement, or can comprise adding the biomaterial and allowingthe native ligament to remain. In preferred embodiments, the umbilicalcord biomaterial is laminated with a load-bearing material, such asplastic sheeting, e.g., TYVEK® prior to folding into the rope-likeconformation.

In another embodiment, the umbilical cord biomaterial can be used torepair a tendon in the hand. In a specific embodiment, for example, theumbilical cord biomaterial is inserted between one or more extensortendons in the hand and bones to provide a gliding surface or a shieldbetween tendon and bone, e.g., to prevent adhesion formation.

Preferably, the umbilical cord biomaterial is stretched prior to repairor replacement of a ligament or tendon. For example, a weight, bearableby the particular piece of biomaterial, can be suspended from thebiomaterial for a time sufficient to allow up to, e.g., about 10%, 15%,or about 20% elongation. Such stretching tends to prevent loosening ofthe biomaterial after application.

The umbilical cord biomaterial, in another embodiment, can also be usedto repair or reinforce a rotator cuff tendon having a defect, e.g., atear. In a preferred embodiment, a piece of the umbilical cordbiomaterial is used to completely cover the rotator cuff tendon defect.

5.2.2 Tympanic Membrane Repair and Other Aural Applications

In another embodiment, the present invention provides methods andcompositions for repair of a tympanic membrane using an umbilical cordbiomaterial. In one embodiment, the present invention provides a methodof repairing a perforated tympanic membrane, comprising contacting saidtympanic membrane with a umbilical cord biomaterial. Said contacting cancomprise shaping a flat piece of the biomaterial into the shape of anentire, or a portion of, a tympanic membrane, and contacting thebiomaterial with the tympanic membrane. In another specific embodiment,said perforation has not healed spontaneously within two months of theappearance of the perforation. As with other applications, thebiomaterial can be contacted with the tympanic membrane while hydrated,or, preferably, while substantially dry (e.g., comprising less than 20%water by weight). The biomaterial can be a single layer, or can be alaminate of two or more layers. In another embodiment, the umbilicalcord biomaterial (whether a single layer or a laminate) contacted withthe tympanic membrane is at least about 70 microns in thickness.

In one embodiment of repairing a tympanic membrane, a tympanic membranehaving a perforation is contacted with an umbilical cord biomaterialsuch that the biomaterial partially or totally occludes the perforation.The perforation to be occluded may be a central perforation, that is, aperforation of any size that does not involve the margin of the tympanicmembrane (i.e., the periphery seated in the auditory canal), or amarginal perforation (i.e., a perforation touching upon, or largelyinvolving, the margin of the tympanic membrane). In another embodiment,only the tympanic membrane is perforated, and no other ear structure isperforated or damaged. In another embodiment, occlusion of theperforation is an adjunct to at least one other surgical procedureinvolving the outer, middle, or inner ear. In another embodiment, therepair of the tympanic membrane is a tympanoplasty. In anotherembodiment, the repair of the tympanic membrane is a myringoplasty.

The benefits of closing a tympanic membrane perforation includeprevention of water entering the ear while showering, bathing orswimming (which could cause ear infection), improved hearing, anddiminished tinnitus. Closure also helps to prevent the development ofcholesteatoma (skin cyst in the middle ear), which can cause chronicinfection and destruction of ear structures.

Tympanoplasty and myringoplasty are generally outpatient procedures. Theotolaryngologist may approach repair of a tympanic membrane perforationeither through the auditory canal (trans-canal approach), or via apost-auricular incision followed by folding the ear forward to exposethe tympanic membrane (post-auricular approach).

Before attempting any correction of the perforation, a hearing test isgenerally performed, and the patient is evaluated for Eustachian tubefunction, as partial or complete loss of Eustachian tube function canexacerbate a tympanic membrane puncture and interfere with the adherenceof a graft to the tympanic membrane. Repair of a perforated tympanicmembrane generally comprises placing an occluding material on themembrane. The patient is evaluated for complications, such as extensionof squamous epithelium through the perforation and into the middle earspace. In such instances, tympanoplasty or myringoplasty is preferablyaccompanied, where possible, by remediation of the complication.

The present invention encompasses repair of a tympanic membrane with anumbilical cord biomaterial either as a first or subsequent therapy. Thatis, the biomaterial may be used to repair a tympanic membrane deformity,such as a perforation, before other remedial measures are tried.Alternatively, repair of a tympanic membrane with biomaterial may beperformed after one or more other remedial measures have been tried andfailed.

In one embodiment, repair of a tympanic membrane with biomaterial mayadditionally comprise applying an anti-infective agent to the graftand/or surrounding ear canal. Thus, in one embodiment, the inventionprovides a method of repairing a tympanic membrane comprising contactingthe tympanic membrane with an umbilical cord biomaterial and ananti-infective agent, e.g., one of the anti-infective agents listed inSection 5.1.2, above. The anti-infective agent can be contacted eitherprior to, concurrently with, or subsequent to contacting the tympanicmembrane with the umbilical cord biomaterial. The anti-infective agentcan be present separate from, or as an integral part of, thebiomaterial. For example, the anti-infective agent can be present on thesurface of the biomaterial, or can be impregnated in the biomaterial. Ina specific example, the anti-infective agent is an antibiotic, abacteriostatic agent, antiviral compound, a virustatic agent, antifungalcompound, a fungistatic agent, or an antimicrobial compound. In aspecific embodiment, the anti-infective agent is ionic silver. In a morespecific embodiment, the ionic silver is contained within a hydrogel.Ionic silver hydrogel is a preferred anti-infective agent because it isbroad spectrum, with no known bacterial resistance; its application andremoval are pain-free, and the hydrogel supports autolytic debridement.In a preferred embodiment, the umbilical cord biomaterial is impregnatedwith silver ions prior to application to the tympanic membrane. Inanother embodiment, the umbilical cord biomaterial is impregnated withsilver ions after application of the biomaterial to the tympanicmembrane, for example, by application of ear drops.

The invention further provides that the use of an umbilical cordbiomaterial to repair a tympanic membrane deformity may be the soletreatment of the tympanic membrane, or may be in addition to anothertherapies or treatment used simultaneously in the course of treating orrepairing a tympanic membrane. For example, the invention provides forthe repair of a tympanic membrane comprising contacting the tympanicmembrane with an umbilical cord biomaterial, and treating the tympanicmembrane using an additional therapy not comprising contacting thetympanic membrane with an umbilical cord biomaterial, where thecontacting and the additional therapy individually or together cause ameasurable improvement in, maintenance of, or lessening of the worseningof, at least one aspect of a tympanic membrane deformity, as compared toa tympanic membrane not contacted with an umbilical cord biomaterial.

The invention further provides for the use of umbilical cord biomaterialto repair an ear condition in conjunction with repair of a tympanicmembrane. For example, the umbilical cord biomaterial can be used toreconstruct or repair the outer or middle ear structures, including theauditory canal and middle ear chamber. The umbilical cord biomaterial,for example, may be used to repair or line the mastoid cavity,particularly where mastoid reconstruction is indicated in addition totympanoplasty. In one embodiment, the umbilical cord biomaterial may beused to line the mastoid cavity where the mastoid cavity comprisesexposed bone, that is, bone with no covering epithelial cell layer. Inanother embodiment, the umbilical cord biomaterial may be used as a ovalwindow graft in stapes surgery, either alone or in conjunction withtympanoplasty or myringoplasty.

5.2.3 Soft Tissue Repair

In another non-limiting embodiment, the invention further provides forthe use of umbilical cord biomaterial to repair a soft tissue injury ordefect in an individual. In one embodiment, the soft tissue defect is anabdominal wall defect. Such an abdominal wall defect, e.g., hernia, isrepaired by suturing one or more sheets of umbilical cord biomaterial,either a single sheet or a laminate of sheets as disclosed elsewhereherein, either dried or hydrated, to the soft tissue such that thedefect is repaired or ameliorated. In a specific embodiment, the softtissue defect is a hernia or abdominal wall defect in which theabdominal wall allows exit of at least part of an organ from theabdominal cavity. In this embodiment, the defect, e.g., discontinuity orhernia, is preferably completely covered with one or more sheets ofumbilical cord biomaterial, whether or not the discontinuity or herniahas been surgically closed. Typically, the umbilical cord biomaterial issutured, stapled, or otherwise fastened to the defect such that repairis effected. In other specific embodiments, said soft tissue defect is adefect in the pelvic floor, an enteroceles, a rectoceles, or acystoceles. In embodiments in which the abdominal wall defect is anopening of the abdominal wall to the exterior of the body, it isgenerally preferred that the defect be contacted with the mesothelialsurface of the umbilical cord biomaterial.

In another specific embodiment, the defect is incontinence, and theumbilical cord biomaterial is used as an adjunct to a suburethral slingprocedure to assist in the repair of gracilis muscle flaps suturedbeneath the urethra.

In another specific embodiment, the soft tissue defect is a leg ulcersuch as, e.g., a venous leg ulcer, arterial leg ulcer, diabetic ulcer ordecubitus ulcer. Repair of a leg ulcer can comprise contacting aportion, or the entirety, of the leg ulcer with one or more pieces ofumbilical cord biomaterial, either a single sheet or a laminate thereof,either dried or hydrated, such that the umbilical cord biomaterialbecomes affixed to the leg ulcer. Typically, the biomaterial becomesaffixed to the leg ulcer without fastening; however, the biomaterial canbe sutured, stapled or glued to the skin surrounding the ulcer, or canbe held in place by, e.g., a bandage or compression boot, or by anyother method known to those of skill in the art.

In another specific embodiment, the soft tissue defect is a surgicaladhesion. In a more specific embodiment, the surgical adhesion is anadhesion resulting from, e.g., gynecological surgery. It is estimatedthat approximately 97% of surgical patients develop adhesions aftersurgery and of these, between 5% and 8% develop complications. Withoutbeing bound by theory, the umbilical cord biomaterial would be aneffective barrier to adhesions in that the amnion epithelial cells onone side of the biomaterials would occupy cell binding sites, making itdifficult for host cells, such as fibroblasts, to attach and penetrate.Thus, the umbilical cord biomaterial can be used to prevent surgicaladhesion by placing the biomaterial between two tissues that wouldordinarily be expected to form a post-surgical adhesion.

In another specific embodiment, the soft tissue defect is a nasal septalperforation. The septal perforation may arise from any cause, e.g.,inherited defect, trauma, drug use, etc. A piece of umbilical cordbiomaterial, suitably shaped, can be placed along the septum in order topartially or completely occlude the perforation, and can be held inplace by one or several sutures or tissue glue.

5.2.4 Ocular Plugs

In another non-limiting embodiment, the invention further provides forthe use of umbilical cord biomaterial in the formation of an ocularplug. An ocular plug at least partially, or, preferably, completely,occludes a hole in, e.g., the sclera, that is, e.g., caused by aninjection, formed as a part of a surgical procedure to, e.g., allowinsertion of a surgical tool into the lumen of the eye; caused bytrauma; etc. Ocular plugs may be configured in any shape to accomplishthe particular purpose at hand, e.g., occluding injection or ocularsurgery-related holes in the sclera, prevention of leakage, drugdelivery, anchoring of the plug, etc.

In one, preferred, embodiment, the invention provides an ocular plugthat comprises a shaft attached to and extending from a cap. Typically,the cap is circular when viewed from the upper face. However, the capmay be oval, square, rectangular, polygonal, irregular, or may appear asa plurality of flanges extending substantially perpendicularly from theshaft. The upper face of the cap distal to the shaft, may behemispherical, curved to a degree other than completely hemispherical,or may be substantially flat. Preferably, the surface of the upper faceof the cap is shaped to approximate the curvature of the eye to promotecomfort and reduce the possibility of inflammation or irritationassociated with the eyelid moving over the face of the cap. The lowerface of the cap proximal to the shaft may be substantially flat, but ispreferably shaped to approximate the curvature of the eye. Preferably,the cap tapers towards the edges so that a smooth transition is madefrom sclera to cap when the eyelid passes over the cap. However, the capneed not taper from center towards the edges, and may have a discerniblyblunt edge.

The cap is preferably of a sufficient diameter to promote seating andmaintenance of position of the plug within the hole in the sclera, andto reduce the possibility of the shaft from passing completely throughthe sclera during or after insertion of the plug into the sclera. Theouter diameter of the cap may be from 1-10 times the diameter of theshaft; preferably, the outer diameter of the cap is between 1-3 timesthe diameter of the shaft.

The shaft, as the remainder of the plug, may be configured to accomplishocclusion of an injection- or ocular surgery-related scleral hole. Theshaft may be thin enough, for example, to occlude the hole made by a 33gauge, or thinner, needle after intravitreous injection, or may be asthick as 1-2 mm in diameter, or more, to occlude holes created during,for example, macular hole surgery. The shaft may be of any sizeappropriate to occlude a particular discontinuity in the sclera. Theshaft is preferably at least as long as a sclera is thick, but may beshorter than the thickness of a sclera, or may be longer. A typicalsclera is 0.35-0.55 mm thick, but may be thicker or thinner. Thethickness depends upon the particular individual, as well as theposition of the discontinuity in the sclera; for example, the scleratends to thin away from the iris and towards the retina. Where the shaftis longer than the thickness of a sclera, the shaft, when the plugcomprising it is fully inserted, projects through the sclera an into thevitreous humor.

The surface of the shaft may be smooth or textured. For example, thesurface of the shaft may be rough, ribbed or knurled so as to enhancecontact between the plug and sclera, thereby reducing the potential forthe plug to work its way out of the scleral hole. Particularly where theplug comprises a cap, the shaft may be ribbed or knurled directionally;that is, ribbed or knurled to promote insertion of the plug into thescleral hole and to discourage passage of the plug in the oppositedirection, i.e., back out of the scleral hole.

In a preferred embodiment, the shaft is substantially cylindrical. Inanother embodiment, the shaft is substantially cylindrical along itsentire length. In other embodiments, the shaft is ovoid, square,rectangular, square or rectangular with rounded edges, polygonal, orirregular in cross-section. In another embodiment, the shaft comprises anarrow portion and a wide portion. Typically, the shaft is attached tothe cap through the narrow portion; the wide portion, distal to the cap,facilitates anchoring of the plug into the sclera. In one embodiment,the cross-sectional area of the wide portion is greater than that of thenarrow portion. The wide portion of the shaft may be manufactured in avariety of configurations. For example, the shaft may flare. Such aflare may be substantially continuous along the length of the shaft, ormay begin at any point along the length of the shaft. In anotherembodiment, the wide portion is a flange or protrusion from a portion ofthe main body of the shaft, e.g., from one side of the shaft. Such aflange or protrusion may have any shape that facilitates maintenance ofthe plug within the scleral hole while not substantially increasing thedifficulty of insertion or the potential for scleral damage duringinsertion. In another embodiment, the wide portion comprises a flange orother protrusion that substantially encircles the shaft. For example,the wide portion may be an inverted cone or frustum, wherein the largerradius of the frustum is wider than the diameter of the shaft. Inanother embodiment, the wider portion of the shaft is a cylinder havinga radius larger than the radius of the shaft. In another embodiment, thewider portion of the shaft has substantially the same cross-sectionalshape as the shaft, but a cross-sectional area larger than thecross-sectional area of the shaft. There is no need, however, for thewide portion of the shaft to have a particular shape relative to thecross-sectional shape of the shaft, and the wide portion need not havethe same cross-sectional shape as the shaft. In another embodiment, theshaft comprises a thread spirally disposed along a portion or all of thelength of the shaft, so that the shaft of the plug functions as a screw.In this embodiment, the thread may proceed clockwise or counterclockwisealong the shaft.

In another embodiment, the plug may be constructed so that the portionof the shaft distal to the cap comprises one or more flaps that may befolded against the shaft during insertion of the plug into the sclera,and which open, or fold away, from the shaft once the flap has beenpushed completely through the sclera. The one or more flaps would act asan anchor.

In one embodiment, the wider portion of the shaft extends into thesclera itself, and serves as an anchor. In another embodiment, part orall of the wide portion of the shaft extends into the vitreous humor.

The end of the shaft distal to the cap may be flat, rounded, or tapered,or may be irregular. The surface of the end may be substantiallyperpendicular to the longitudinal axis of the shaft, or may be tilted,giving the end of the shaft a barbed appearance.

In one embodiment, the ocular plug comprises an opening extending atleast the portion of the cap distal to the shaft, and, optionally, intothe shaft. The opening can be used, for example, to receive a wire offixed gauge. The wire is used to pick up the ocular plug and guide theocular plug into a scleral discontinuity.

In another embodiment, the ocular plug does not comprise a cap. Forexample, the ocular plug may comprise a shaft only. In this embodiment,the shaft may be formed in any of the configurations as for the shaftsof a plug with a cap as discussed above. For example, in its simplestform, the plug may simply be a cylinder, with a smooth, ribbed, knurledor textured surface, or may comprise one or more wide portions that canact as anchors. In one embodiment, the shaft (that is, the plug)comprises two wide portions. In a specific embodiment, the shaft isdumbbell-shaped. The dumbbell shape may be accomplished, for example, bythickening the ends of the shaft so that the change in thickness fromcenter of the shaft to either end is continuous; alternatively, thechange in thickness from center of the shaft to either end isdiscontinuous. Preferably, in this embodiment, the length of the shaftbetween the wide portions (e.g., the ends of the dumbbell) is at leastthe thickness of the sclera.

Ocular plugs may be pre-made to standard sizes, or may be custom-made tofill particular scleral holes or discontinuities, whether anticipated(as in the case of surgery) or unanticipated. In one embodiment,therefore, the invention provides an ocular plug, wherein said ocularplug has a shaft of a reproducible, standard size. The ocular plug mayalso be custom-made for a particular discontinuity. In specificembodiments, the standard or custom-made diameter size of a shaft forsaid ocular plug is a diameter sufficient to substantially occlude ascleral hole caused by passage of a 33, 32, 31, 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or larger gaugeneedle. In other specific embodiments, the standard or custom-madediameter of a shaft for said ocular plug is about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 millimeters, or wider. In otherspecific embodiments, the standard length of the shaft of said plug is0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90,0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45 or 1.50millimeters.

Ocular plugs may be made from umbilical cord biomaterial by any methodused to create or produce molded devices. Preferably, the umbilical cordbiomaterial used to make an ocular plug is a dried, decellularizedbiomaterial.

Ocular plugs may be made, for example, by stamping the plugs from asheet of the biomaterial using a shaped stamp. Alternatively, the plugsmay be cut from a sheet of the biomaterial, or may be formed by removalof unwanted material from a block of the biomaterial. In a preferredembodiment, an ocular plug is formed using a mold. Where the ocular plugis formed using a mold, the biomaterial is preferably first made into aliquid, slurry, paste, or similar material amenable to forming in amold.

In an exemplary embodiment of a method of making the ocular plug,umbilical cord biomaterial is first reduced to a collection ofparticles; that is, the biomaterial is micronized. The biomaterial maybe micronized to a particle size of anywhere from about 1 micron toabout 1 millimeter. Generally, the larger the particle size, the moreporous the plug. Any method may be used to micronize the biomaterial,for example, ultrasound, physical shearing, homogenization, etc. Suchmicronization may be done dry (that is, by micronizing the biomaterialwithout any additional liquid), or may be done using a micronizationliquid or carrier. If micronization with a liquid is performed, theliquid may be any physiologically acceptable liquid or solution thatdoes not significantly degrade the biodegradable material, e.g., thetertiary structure of the proteins comprising the biomaterial.Typically, the ratio of biomaterial to liquid is 25 mg/ml to 300 mg/ml,but more or less of the biomaterial may be used. Determination that adesired particle size has been achieved may be accomplished by any meansknown in the art, e.g., microscopic examination, comparison to bead sizestandards, etc.

Once the desired micronized biomaterial is obtained, whether in wet ordry form, the micronized biomaterial is injected or otherwise forcedinto the mold and allowed to set. In one embodiment, the wet micronizedbiomaterial is forced into the mold and is then frozen, e.g., at atemperature of from −5° C. to −160° C. (though higher or lowertemperatures would also work) for a time sufficient to allow icecrystals to form and grow, e.g., 2 hours to several days. The frozenplug is then freeze dried to substantial dryness, that is, to a watercontent of about 20% by weight or less. Preferably, any plugs formedusing the biomaterial are freeze-dried to substantial dryness.Freeze-drying is particularly preferred as the process allows for thedevelopment of pores in the biomaterial constituting the plug. Plugs mayalso be heat-dried, but heat applied to dry the plugs is preferably notheat that would cause the breakdown of any component of the biomaterial.For example, in various embodiments, an ocular plug formed frombiomaterial may be dried at about 70° C., about 65° C., about 60° C.,about 55° C., about 50° C. or about 45° C., or less than about 70° C.,less than about 65° C., less than about 60° C., less than about 55° C.,less than about 50° C. or less than about 45° C.

Once the plugs are freeze-dried, or dried by other method, thebiomaterial is preferably cross-linked to provide mechanical stabilityand integrity. Crosslinking may be accomplished by any method known inthe art; particularly preferred are radiation, chemical, or heatcrosslinking. Radiation crosslinking is preferred. Radiation used may beany known in the art to be useful for such a purpose, for example,electron-beam or e-beam radiation, gamma radiation or ultravioletradiation. E-beam radiation is preferred. See, e.g., Odland, U.S. Pat.No. 5,989,498 “E-beam Sterilization of Biological Materials,” Theintensity of radiation used may be that ordinarily used for thesterilization of medical instruments. The biomaterial may also bechemically crosslinked using any chemical crosslinking methodology knownin the art, for example, thiol-thiol crosslinking, amide-amidecrosslinking, amine-thiol crosslinking, amine-carboxylic acid andthiol-carboxylic acid crosslinking, etc., as appropriate for thematerial from which the plug is made. The plug may also be heatcrosslinked, typically using a thermal dehydration process. Mostpreferably the heat used for such crosslinking does not significantlydegrade or structurally weaken the material in any way. Plugs may beheat crosslinked, for example, by placing the plugs in a vacuum oven at105° C. for 1-5 hours, or until the desired structural integrity ordegree of crosslinking is achieved.

5.2.5 Other Uses

The umbilical cord biomaterial can be used in any other medicalapplication in which like materials are used, e.g., as a patch for woundrepair.

In one embodiment, the umbilical cord biomaterial is used as a wrappingor covering for a replacement eye orb, e.g., a hydroxyapatite sphere.

In another embodiment, the umbilical cord biomaterial can be used torepair or ameliorate a cardiac defect. Such defects include, but are notlimited to, cardiac wall defects, areas of necrosis due, e.g., toischemia; repair of a cardiac valve; repair of patent foramen ovale, andthe like. In another embodiment, the umbilical cord biomaterial isseeded or inoculated with cardiomyocytes. Immediately, or followingculture to allow proliferation of the cardiomyocytes, the umbilical cordbiomaterial is implanted into a cardiac defect as a living patch. Inpreferred embodiments, the umbilical cord biomaterial is placed so as tocover the defect completely.

5.3 Kits

The present invention further provides kits comprising one or morepieces of umbilical cord membrane biomaterial. Preferably, each of theone or more pieces the biomaterial is individually sterilely wrapped,e.g., in a peel-pouch.

In a more specific embodiment, the kit comprises a piece of umbilicalcord biomaterial that is at least about 2×8 cm. In another embodiment,said kit comprises a piece of umbilical cord biomaterial approximatelythe size of an eardrum. The kit may comprise one or more pieces ofumbilical cord biomaterial and any other medical device, disposable ordrug that would facilitate treatment of a disease, disorder or conditiontreatable using the biomaterial. Preferably, each piece of the umbilicalcord biomaterial in the kit is provided as a single sheet or patch in asterile container or wrapping separate from the remainder of kitcontents. In another embodiment, the kit comprises two or more pieces ofumbilical cord biomaterial, separately wrapped or contained. In anotherembodiment, said kit comprises a support for the umbilical cordbiomaterial. In specific embodiments, the support may be a natural or asynthetic material. In other specific embodiments, said support is aplastic film, plastic sheet, or a stretchable plastic wrap. In anotherembodiment, said kit comprises one or more disposables, e.g., papertissues or towels, mats, cotton swabs, plastic or rubber gloves,disposable forceps, or the like. In a specific embodiment, saiddisposables are bandages, means for sterilizing skin, swabs, gloves, orsterile sheets. In another embodiment, said kit comprises ananti-infective agent, for example, an antibiotic ointment, cream, orspray. In another embodiment, said kit comprises a piece of umbilicalcord biomaterial and one or more wound healing agents. In a specificembodiment, said wound healing agent is PDGF, TGF, hyaluronic acid,fibrin, or fibronectin. In another embodiment, said kit comprisesumbilical cord biomaterial and a means for applying compression to apart of the body. In a specific embodiment, of any of the kits above,the kit comprises an instruction sheet suitable for use by a non-medicalend user; an instruction sheet suitable for use by an end user in amedical profession; or a materials safety data sheet; or a combinationthereof.

6. EXAMPLES 6.1 Example 1 Production of Umbilical Cord Biomaterial

The following example demonstrates one method of preparing umbilicalcord biomaterial.

Materials and Equipment

The following items were obtained and, where appropriate, sterilized:human placenta (less than 48 hours old at the start of processing);surgical clamps/hemostats; scissors; scalpels; tweezers; Halstedmosquito; Adson bayonet forceps; grooved directors; cell scraper;autoclaved gauze; stainless steel rinsing trays; stainless steel cups;stainless steel processing trays. 0.9% NaCl solution; sterile water;specimen containers; personal protective equipment (including sterileand non-sterile gloves); certified clean room; decellularizing solution(0.5% deoxycholic acid solution); rocking platform (VWR Model 100);timer (VWR TRACEABLE° model); disinfected silicone grid; PVC wrap film;vacuum pump (Schuco-Vac 5711-130); heat dryer (BioRad Model 583);sterile cutting board; pouches for packaging (COT-360, 361, 362);stainless steel ruler; TRACEABLE° Digital Thermometer (Model 61161-364,Control Company); Accu-Seal Automatic Sealer (Accu-Seal, Model 630-1 B6or 730-16B) with air compressor; and waterproof resealable bags(CCT-03S).

Procedure

A sterile field was set. The placenta was removed from the transportcontainer and placed into a sterile stainless steel tray. Using surgicaldamps and scissors, the umbilical cord was cut off approximately 2inches from the placental disc. The umbilical cord was rinsed withsterile 0.9% NaCl solution as many times as necessary to remove as muchblood as possible; optionally, fingers were used to squeeze remainingblood from vessels. The umbilical cord was optionally placed in aseparate sterile container cup prefilled with sterile 0.9% NaClsolution, if the cord did not have to be processed immediately. Theharvested umbilical cord was placed in a refrigerator at 4° C. untiluse. The placental disk was placed back into the transport container tobe utilized for other projects, or discarded.

The umbilical cord was processed as follows. The umbilical cord wasremoved from the specimen container, and squeezed to remove anyremaining blood from vessels prior to introducing the umbilical cord toa processing tray. The umbilical cord was placed into a sterilestainless steel processing tray, and cut into segments 12 to 15 cm inlength. The umbilical cord vein was then located for each segment, andcanalized using an Adson bayonet forceps or grooved director. The veinand umbilical cord were then cut longitudinally, using scissors, untilboth the vein and umbilical cord were fully open. The umbilical cord,was placed on the processing tray with the opened vein side facingupward. The umbilical cord and vein were then bluntly dissectedlongitudinally between the vein wall and the umbilical cord wall withsterile tweezers or mosquito clamps. When both sides were separated, thevein was carefully removed. After vein removal, the two arteries werelocated and removed in the same manner. Depending on the purpose of thestudy, the resulting umbilical cord membrane (biomaterial) was placed ineither in saline solution or in 1% deoxycholic acid solution (adecellularizing solution) and stored at 4° C. until serological testingresults become available.

Storage and Quarantine of Umbilical Cord Membrane

The umbilical cord membrane, obtained as outlined above, was kept insterile 0.9% saline solution or 1% deoxycholic acid solution for 10-20days at 4° C. until serological test results, if ordered, wereavailable. Saline solution, where used, was changed every 3 days. 1%deoxycholic acid solution, when used, was changed every 5 days.

Umbilical Cord Membrane Cleaning and Rinsing

A sterile field was set with a new set of sterilized trays as above. TheUmbilical cord membrane was removed from the refrigerator and placedinto a stainless steel processing tray. Sterile 0.9% saline solution isadded to cover the bottom of the tray. All, or substantially all,residual deoxycholic solution, where used, was removed, and remainingcells and debris were removed from both sides of the tissue using a cellscraper and sterile tweezers. Sterile 0.9% saline solution was used asneeded to aid in removal of the cells and debris. The umbilical cordmembrane was rinsed three times in a separate stainless steel rinsingtray filled with sterile 0.9% saline solution. The saline solution waschanged between each cleaning step. The umbilical cord membrane was thenplaced into a new sterile specimen container containing about 150 mLsaline solution, and placed on a rocking platform for agitation for 5minutes at setting #6. The scraping and rinsing steps were repeated onceas necessary. The umbilical cord membrane was then placed into a sterilespecimen container containing 150 mL sterile water, and placed on arocking platform for agitation for 20 minutes at setting #6. Thisrinsing step was repeated three times.

Drying the Umbilical Cord Membrane

A TYVEK® sheet was placed onto a stainless steel processing tray. Thecleaned umbilical cord membrane segments were removed from the specimencontainer one piece at a time, and excess fluid was gently squeezed out.The membrane segments were then placed on the surface of the TYVEK®sheet, epithelium side up, and gently stretched until flat. The membranewas then dried at about 50° C.±1.0° C. in a vacuum dryer. Sterile gauzewas placed on the drying platform of the vacuum dryer, covering an areaslightly larger than the area of the TYVEK® sheet. The total thicknessof the gauze layer did not exceed the thickness of one folded 4×4 gauze.A sheet of silicone framing mesh was placed on top the gauze, smoothside up. The TYVEK® sheet with the tissue was then placed on the heatdryer platform on top of the silicone mesh. Another TYVEK® sheet wasthen placed on top of the tissue. A piece of PVC wrap film was then cutlarge enough to cover the entire drying platform, and pulled so that thefilm pulled tightly against the TYVEK® sheet (that is, was “sucked in”by vacuum) and so that there were no air leaks and no wrinkles over thetissue area). The vacuum pump was then set to approximately −22 inchesHg, and heat/vacuum drying was allowed to proceed for a total of about120 minutes. Approximately 30-45 minutes into the drying process, thesterile gauze layer was replaced.

A new sterile field was set with a sterilized drying kit and cuttingboard. With the pump still running, the plastic film was removed fromthe TYVEK®, and the sheet and tissue were placed on a cutting board withthe epithelium side of the tissue facing upward. The dried membrane (nowumbilical cord biomaterial) was then gently removed from the TYVEK®sheet. The biomaterial segments were then cut with a scalpel intosegments of a specified size, typically 2×2 cm or 1×1 cm. The dried,sized umbilical cord biomaterial was then placed and sealed into apeel-pouch package.

6.2 Example 2 Production of Umbilical Cord Biomaterial Laminate

Objective: To increase the size of a sheet of umbilical cord biomaterialfor hernia repair.

Materials and Methods: All cited dimensions are approximate. Umbilicalcord membrane from a 23.5 hour-old placenta was collected and processedas in Example 1 up to the point of drying. The final size of themembrane was approximately 35 cm by 4 cm. The membrane was cut intothree pieces approximately 10 cm long. The pieces were arranged so as tooverlap by about 2 cm on the long edge, and were dried at 50° C. betweentwo sheets of TYVEK®.

Results: The biomaterial comprising laminated membrane thus obtained wasapproximately 10 cm by 10 cm. The sections did not separate uponrehydration in saline for 72 hours.

Umbilical cord membrane can also be laminated by placing two or morepieces of the biomaterial, interior (of the umbilical cord) side down,on a substrate in a mounting frame. The laminated membrane is thenplaced in a gel dryer and dried to substantial dryness (≦about 20% watercontent by weight) to produce a laminated umbilical cord biomaterial.

Another method of constructing a thicker biomaterial is to laminateintact umbilical cord (including Wharton's jelly but lacking arteriesand vein). The intact cord is then processed, e.g., by rinsing, soakingin a solution such as a buffered saline solution, e.g., phosphatebuffered saline, or a mild ionic or nonionic detergent solution. Thecord is dried in a vacuum dryer to create an intact, double-layerbiomaterial. Two or more layers of this double-layer material can belaminated by layering the biomaterials and drying further in a heatedvacuum dryer. The drying/dehydration process can be heat drying or anyother processes as described below.

6.3 Characterization of Dried Umbilical Cord Biomaterial

A study was undertaken to examine biomaterial made of heat dried humanumbilical cord membrane (HUC) after sterilization by different doses ofgamma irradiation. Samples of HUC were sterilized with 0, 20, 25, 30, or40 kGy and then examined for water uptake (mass and thickness change),denaturation temperature, and tensile mechanical properties. HUC sampleshad been incubated for either 10 or 20 days in 1% D-cell (deoxycholicacid) solution during preparation.

6.3.1 Water Uptake

Individual samples of HUC for each condition (n=3) were weighed on amicrobalance. Samples were then incubated in 10 mL of phosphate bufferedsaline at 37° C. for 1 hour. Samples were removed from the PBS andblotted dry a minimum of three times with a KIMWIPE® tissue. The sampleswere again weighed on a microbalance. The percentage water uptake ([wetweight (Ww)−dry weight (Wd)]/Wd*100) and equilibrium water content([Ww−Wd]/Ww*100) were calculated.

FIG. 1 summarizes the results of the rehydration for HUC incubated for10 and 20 days. The initial water uptake of the control (nonsterilizedsamples) was much higher for HUC incubated for 20 days than for HUCincubated for 10 days, possibly due to the loosening of the membraneproteins by the detergent effect of the deoxycholic acid in the D-cellsolution. Water uptake and equilibrium water content closely matched forthe 10 and 20 days samples that were sterilized at all radiation doses.There was a linear decrease in the water uptake and the equilibriumwater content of both sets of samples with increasing radiation dose.Even at the highest radiation dose, the membranes took up at least theirown weight in water.

6.3.2 Changes in Thickness

Individual dog bone shaped (see, e.g., FIG. 4) samples of HUC for eachcondition (n=8) were mounted in squares of vellum paper so that themembrane could be easily handled during and after hydration. Thethickness was measured in three locations for each sample and averaged.Samples were then incubated in 10 mL of phosphate buffered saline at 37°C. for 1 hour. Thickness measurements were repeated after hydration.

Overall, the average dry thickness of the HUC was ˜70 μm, with the 10and 20 day samples, having average thickness of 57 and 86 μmrespectively (Table 1 and Table 2). There appeared to be no correlationbetween radiation dose and dry thickness of the HUC. After rehydration,there was a marked difference in thickness between the sterilized andnon-sterilized samples. There was little difference in the rehydratedthickness between the 10 and 20 day samples. Without wishing to be boundby any theory or mechanism, the difference observed can be due tocross-linking of collagen molecules caused by irradiation. Thereappeared to be a decrease in the magnitude of the thickness change uponrehydration with increasing dose; this effect was more pronounced withthe 10 day samples.

TABLE 1 Changes in thickness of 10 day incubated HUC during hydrationDose increase (KGy) dry (um) SD wet (um) SD (%) SD 0 61 23 243 82 316%109%  20 44 10 107 20 147% 19% 25 58 24 132 40 134% 28% 30 67 22 125 45 87% 32% 40 57 23 106 46  91% 36%

TABLE 2 Changes in thickness of 20 day incubated HUC during hydrationDose increase (KGy) dry (um) SD wet (um) SD (%) SD 0 88 22 279 51 240%123%  20 76 22 149 26 106% 48% 25 85 50 130 21  83% 61% 30 61 23 138 51125% 31% 40 121 32 209 46  77% 36%

When the change in thickness of the membranes was compared to the wateruptake (FIG. 2), there was a loose correlation between the amount ofwater taken up and the increase in thickness. The magnitude of the wateruptake and the change in thickness both decreased with increasing gammaradiation dose. There was a stronger correlation between water uptakeand thickness change for samples incubated for 10 days than thoseincubated for 20 days. This is due partially to the fact that thesamples incubated for 20 days showed less difference between samples,but greater variability within a set of samples.

6.3.3 Denaturation Temperature

Individual samples of HUC for each condition (n=3) were incubated in 10mL of phosphate buffered saline at 37° C. for 1 hour. Samples wereremoved from the PBS and blotted dry a minimum of three times with aKIMWIPE® tissue. The samples were sealed in aluminum hermeticdifferential scanning calorimeter (DSC) pans and tested in a TAInstruments modulated DSC (Q1000) in standard mode from 5-110° C. at 10°C./min. TA Instruments' “Universal Analysis” software was used tocalculate the onset and peak values of the denaturation point of themembranes.

FIG. 3 graphically summarizes the denaturation temperature results forthe HUC samples. The results are identical for samples incubated for 10and 20 days. Onset and peak temperatures were only a few degreesdifferent for each of the samples, and both onset and peak temperaturesdecreased linearly with increasing radiation dose. There was very lowvariability in the results.

Time of incubation in D-cell solution did not affect the denaturationtemperature. There was a linear decrease in the onset and peakdenaturation temperatures with increasing radiation dose for both the 10and 20 day samples. From the denaturation data, there appeared to be nodifference between soaking HUC for 10 or 20 days in 1% D-cell solution.

6.3.4 Tensile Properties

Individual dog bone shaped samples of human umbilical cord membrane foreach condition (n=8) were mounted in squares of vellum paper so that themembrane could be easily handled during and after hydration. Sampleswere then incubated in 10 mL of phosphate buffered saline at 37° C. for1 hour. The tensile properties of the membranes were evaluated based onAmerican Society for Testing and Materials protocol D1708 (ASTM D1708);see FIG. 4 for more details of testing. Samples that had been cut alongthe long axis of the cord (longitudinal sections) and samples that hadbeen cut perpendicular to the long axis (cross section) were testedwhere samples were available for each condition.

No differences were found between 10 and 20 day samples. Tests on theunsterilized human umbilical cord membrane determined the tissue to beanisotropic, with the tensile properties being different depending onthe direction in which the samples were oriented (Table 3). Longitudinalsamples had a higher stress at break and a higher modulus than crosssection samples, and cross sectional samples had a greater range ofvalues for the extension at break (how far the tissue stretched beforebreaking). In all cases, when the load was removed from the unsterilizedsamples, the tissue appeared by visual inspection to return to itsoriginal shape with no obvious distortions. Force was applied at a rateof 33 mm/min.

TABLE 3 Tensile testing results for non-sterilized HUC (10 and 20 daysamples) Stress Extension at break Modulus at break Orientation (MPa)(MPa) (%) Longitudinal 2.3-8.8 14.2-27.5 135-270 Cross 0.1-3.5 0.5-8.9 60-480

While there were no differences between the tensile properties of the 10and 20 day samples or the different radiation doses, there were strikingdifferences in how unsterilized and sterilized samples failed.Nonsterilized samples failed by breakage of the sample, while thesterilized samples failed mostly by delamination of layers of tissue.Additionally, the sterilized tissue was noticeably deformed after theload had been removed.

The tensile properties of HUC most closely resembled skin (Table 4).This was true of both the sterilized and non-sterilized tissue.

TABLE 4 Comparison of tensile properties of HUC (10 and 20 day samples)and other tissues Tensile Tensile Extension strength modulus at break(MPa) (MPa) (%) Tendon/ligament   100-2,000 50-150  5-50 Articularcartilage 1-10 Skin 10-40 2-20  50-200 Compact bone 10,000-20,000 2-3HUC (long) non-sterile 2.3-8.8 14.2-27.5  135-270 HUC (long) (sterile) 1.1-12.4 7.4-51.4  60-258

6.3.5 Suture Pull-Out Strength

The umbilical cord biomaterial was determined to have a superior suturepull-out strength compared to dried human amniotic membrane. In a testsimilar to that described in Section 6.3.4, above, one short side of a1×2 section of umbilical cord biomaterial was glued to vellum paper, andthe other short side sutured to a second piece of vellum paper, asdepicted in FIG. 5A. The pieces of vellum paper were held by grips, anda load was applied to the suture at a rate of about 12.7 mm/min. Theumbilical cord biomaterial demonstrated an average pull-out resistanceof about 1.4 Newtons (N), with a range of about 0.75 N to about 2.4 N,while the dried amniotic membrane demonstrated a pull-out resistanceaveraging about 0.3 N. See FIG. 5B.

6.4 Example 4 Biocompatibility of Umbilical Cord Biomaterial

This study evaluated the host response to an implant made of umbilicalcord biomaterial during absorption following subcutaneous implantationin a rat model.

6.4.1 Materials and Methods

Test materials for implantation consisted of umbilical cord biomaterialor high density polyethylene (HDPE; control). Umbilical cord biomaterialwas provided as dried umbilical cord membrane measuring approximately 1cm×1 cm sections prepared in either 0.9% NaCl (Test article A;non-decellularized) or 1% deoxycholic acid (Test articles B and C;decellularized).

The 16 rats used in the experiments were ten week old male Rattusnorvegicus strain H1A®:(SD)CVF® (Hilltop Lab Animals, Inc.). Animalweight at the time of implantation ranged from 344 grams to 392 grams.Maintenance of animals during the experiment conformed to StandardOperating Procedures based on the “Guide for the Care and Use ofLaboratory Animals”.

On the day of the implant, each rat was identified and weighed. Groupsof four animals were arbitrarily assigned to be terminated 1 week, 3weeks, 6 weeks or eight weeks after implantation (Table 5).

TABLE 5 Implantation of test articles: Bilateral Animal ImplantationTermination Number Left Right Interval 1 C A 1 week 2 C A 3 B A 4 B A 5C A 3 weeks 6 C A 7 B A 8 B A 9 C A 6 weeks 10 C A 11 B A 12 B A 13 C A8 weeks 14 C A 15 B A 16 B A

For implantation, the animals were anesthetized by intraperitonealinjection of ketamine hydrochloride and xylazine (66 mg/kg and 9 mg/kg,respectively) dosed at 2.25 ml/kg. The implant region was scrubbed witha germicidal soap and wiped with 70% alcohol. Separate incisions weremade on each side of the back through the skin and parallel to thelumbar region of the vertebral column. A pocket was formed by bluntdissection in the subcutaneous tissue on each side of the back. Onesection of the test material was implanted into each pocket such that itlay as flat in the pocket as reasonably possible. A nonsorbable suturewas cut into approximately 1 cm length sections and placed at each testarticle implantation site as a location marker. One section of thenegative control article (HDPE) was similarly implanted caudally to thesections of the test article. The skin was closed with wound clips.

Following implantation, the animals were observed daily for generalhealth, and the incisions were examined for adverse reactions untilwound clip removal. Detailed examinations for clinical signs of diseaseor abnormality were conducted weekly and at termination.

At 1, 3, 6 and 8 weeks after implantation, the designated animals wereweighed and euthanized by carbon dioxide inhalation. Macroscopicobservation of the viscera was conducted. The general appearance of theskin at the implantation sites was recorded. The implant sites wereexposed by incision along the midline from the proximal to the distalend of the rat, and the skin was gently pulled away from theimplantation site. The implanted materials were measured to the nearestmillimeter (length and width) and the color and consistency of thesurrounding tissue was documented. The sites were also photographed. Theimplant sites and any abnormal tissues were excised and preserved in 10%neutral buffered formalin (NBF) until further processing. Afterfixation, the implant sites and any abnormal tissues were histologicallyprocessed (embedded, sectioned and stained with hematosylin and eosin)for microscopic evaluation by a pathologist.

Implantation sites were evaluated to assess any change in the integrityof the form of the test material. The local tissue response wasevaluated and compared to the reactions at the negative control articlesited. The evaluation included characterization of the test material inregard to acute inflammation, chronic inflammation, granulation tissueformation, foreign body reaction, and foreign body giant cell formation.In addition, the formation and the thickness change of the fibrouscapsule around the implants, the change in implants' characteristics atdegradation (e.g., size and shape, formation of particles, fibers andamorphous gel, etc.) were also evaluated. Microscopic cellular changeswere graded according to severity on a scale of 0 to 4.

6.4.2 Results

Clinical observations. All animals appeared clinically normal throughoutthe study, Minor scabs or ulcerations were noted at the anestheticinjection sites; these areas resolved without treatment.

Body weight data. In general, all rats gained weight over the course ofthe study, and weight gains were considered acceptable.

Macroscopic observations. Generally, all animals, and the appearance ofthe skin, appeared macroscopically normal following termination. Ingeneral, the color and consistency of the tissue surrounding the implantappeared normal for all animals from three week termination interval. Atthe one week termination interval, some signs of surgical trauma wasstill evident, which is typical. For each animal, symptoms arising fromimplantation of the test articles (umbilical cord biomaterial) were mildenough that the biomaterial was considered a nonirritant.

Implant absorption. Implant size at the various termination points ispresented in Tables 6 and 7. In several cases, the implant was found tohave been completely absorbed by the host. Average implant sizes for thetest articles at each of the terminations points is shown in Table 8.

TABLE 6 Measurements of implanted materials at termination - left sideAnimal Length Presence/Absence Group # Site (mm) Width (mm) of TestMaterial 1 Week 1 Test 9.8 7.8 Present Control 11.8 10.0 Present 2 Test8.6 8.9 Present Control 13.5 10.7 Present 3 Test 10.5 10.9 PresentControl 13.8 10.7 Present 4 Test 10.0 8.8 Present Control 14.1 11.4Present 3 Weeks 5 Test 5.3 8.3 Present Control 11.0 13.6 Present 6 Test4.2 6.2 Present Control 12.2 15.6 Present 7 Test 7.2 5.0 Present Control10.9 13.1 Present 8 Test 8.2 7.6 Present Control 11.4 14.0 Present 6Weeks 9 Test 6.9 6.9 Present Control 8.9 10.9 Present 10 Test 5.9 4.6Present Control 10.5 13.5 Present 11 Test 6.1 6.2 Present Control 10.611.8 Present 12 Test 6.2 6.9 Present Control 11.4 10.5 Present 8 Weeks13 Test 8.4 6.0 Present Control 12.1 12.1 Present 14 Test NA NA AbsentControl 14.8 12.0 Present 15 Test NA NA Absent Control 12.7 14.0 Present16 Test 9.3 8.1 Present Control 12.5 13.5 Present

TABLE 7 Measurements of implanted materials at termination - right sideAnimal Length Presence/Absence Group # Site (mm) Width (mm) of TestMaterial 1 Week 1 Test 10.4 9.9 Present Control 12.2 12.2 Present 2 Test7.5 10.1 Present Control 12.1 11/2 Present 3 Test 7.8 6.8 PresentControl 10.8 11.6 Present 4 Test 8.3 6.6 Present Control 11.7 12.2Present 3 Weeks 5 Test 5.5 8.0 Present Control 12.3 13.2 Present 6 Test6.1 5.5 Present Control 11.5 10.9 Present 7 Test NA NA Absent Control10.7 10.2 Present 8 Test 7.6 5.5 Present Control 10.3 11.7 Present 6Weeks 9 Test NA NA Absent Control 10.6 9.5 Present 10 Test NA NA AbsentControl 10.4 14.9 Present 11 Test NA NA Absent Control 11.4 10.5 Present12 Test NA NA Absent Control 10.2 12.7 Present 8 Weeks 13 Test 7.8 5.6Present Control 12.3 11.6 Present 14 Test 11.2 5.9 Present Control 14.412.6 Present 15 Test NA NA Absent Control 13.5 12.2 Present 16 Test NANA Absent Control 11.7 13.8 Present

TABLE 8 Average length and width of test articles Average Length andWidth of the Test Articles 4830 4825 4838 Length Width Length WidthLength Width Interval (mm) (mm) (mm) (mm) (mm) (mm) 1 weeks 9.2 8.4 10.39.9 8.5 8.4 3 weeks 4.8 7.3 7.7 6.3 4.8 4.8 6 weeks 6.4 5.8 6.2 6.6Absorbed Absorbed 8 weeks 4.2 3.0 4.7 4.1 4.8 5.8

Further results indicated that both decellularized andnon-decellularized umbilical cord biomaterial have excellentbiocompatibility. Animal responses to both biomaterials was similar tocontrol USP grade HDPE. No fibrous tissue encapsulations developedduring the course of the implantation study, whereas HDPE implantsdeveloped fibrosis at later stages of the study. While bothdecellularized and non-decellularized biomaterials were biodegradable,it was noted that implanted decellularized membranes lasted longer(e.g., about 6 to 8 weeks) than non-decellularized biomaterials (about 3to 6 weeks).

EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1. An umbilical cord-derived biomaterial comprising an isolatedmammalian umbilical cord membrane and Wharton's jelly, wherein saidbiomaterial comprises less than 20% water by weight.
 2. The biomaterialof claim 1 that comprises at least one umbilical vessel.
 3. Thebiomaterial of claim 1 that is decellularized.
 4. The biomaterial ofclaim 1 comprising an exogenous bioactive molecule.
 5. The biomaterialof claim 4, wherein said bioactive molecule is a cytokine or a growthfactor.
 6. The biomaterial of claim 4, wherein said bioactive moleculeis an extracellular matrix protein.
 7. The biomaterial of claim 6,wherein said extracellular matrix protein is collagen, fibronectin,elastin, vitronectin, or hyaluronic acid.
 8. The biomaterial of claim 4,wherein said bioactive molecule is hyaluronic acid.
 9. The biomaterialof claim 8, wherein said hyaluronic acid is crosslinked to saidumbilical cord membrane.
 10. The biomaterial of claim 1, comprising anexogenous polymer.
 11. The biomaterial of claim 10, wherein saidexogenous polymer is a synthetic biodegradable polymer or an anionicpolymer.
 12. The biomaterial of claim 4, wherein the bioactive moleculeis an antibiotic, a hormone, a growth factor, an anti-tumor agent, ananti-fungal agent, an anti-viral agent, a pain medication, ananti-histamine, an anti-inflammatory agent, an anti-infective agent, awound healing agent, a wound sealant, a cellular attractant, ascaffolding reagent, or a small molecule.
 13. The biomaterial of claim12, further comprising a hydrogel composition.
 14. The biomaterial ofclaim 1, additionally comprising an exogenous stem cell.
 15. Thebiomaterial of claim 3, additionally comprising an exogenous stem cell.16. The biomaterial of claim 14, wherein said exogenous stem cell is aplacental stem cell, a mesenchymal stem cell, an embryonic stem cell, ora somatic stem cell.
 17. The biomaterial of claim 16, wherein saidsomatic stem cell is a neural stem cell, a hepatic stem cell, apancreatic stem cell, an endothelial stem cell, a cardiac stem cell, ora muscle stem cell.
 18. A laminate comprising a plurality of layers,wherein at least one of the layers comprises the biomaterial of claim 1.19. A method of producing a biomaterial comprising isolating anddecellularizing a composition comprising an umbilical cord membrane. 20.A biomaterial made by the method of claim
 19. 21. A method of deliveringa therapeutic agent to a subject comprising contacting the subject withthe composition of claim 1, wherein said composition comprises atherapeutic agent.
 22. A method of repairing a tympanic membrane havinga deformity, comprising contacting said tympanic membrane with abiomaterial comprising an isolated mammalian umbilical cord membrane.